JP6862497B2 - NME inhibitors and methods of using NME inhibitors - Google Patents

Description

The present invention relates to inhibitors of the NME family of proteins. The present invention further relates to a method of using an inhibitor.

In recent years, anti-cancer agents, which are cytotoxic substances, have been replaced or increased as "high-performance" drugs that directly or indirectly target special molecules that promote cancer cell growth. Ideally, the targeted molecule is more expressed in cancer cells than in normal cells. More preferably, it is a drug that targets a molecule that is expressed almost exclusively in cancer cells or cancer tissues but not in healthy human adult tissues. In that case, the target molecule is effectively incapacitated without significantly damaging the patient's healthy tissue.

We have previously reported the discovery that the NME protein is a ligand for the MUC1 * growth factor receptor and that these ligand receptor pairs mediate the growth of both stem and cancer cells (Mahanta et al,). 2008, Hikita et al, 2008, Smagghe et al, 2013). Prior to that, NM23-H1 and NM23-H2 (NME1 and NME2) were treated as having a role in differentiation, but were filled with inconsistent reports (Lombardi et al, 1995). .. First, NM23 has been identified as an inhibitor that prevents leukemic cells from reaching final differentiation, a proof of disease (Okabe-Kado, J., et al. 1985, Okabe-Kado). , J., et al. 1992, Okabe-Kado, J., et al. 1995).
However, prior to disclosure by the present inventors, NM23-H1 and H-2 are ligands for the MUC1 * growth factor receptor that promotes stem and cancer cell growth through lingand-induced dimerization of the MUC1 * extracellular domain. It was not known why NM23 had to be a dimer in order to be differentiated or active, or it was significantly involved in dimerizing its target receptor. .. We have found that the dimer NM23 binds to and dimerizes MUC1 * in cancer and stem cells, and promotes cancer growth and survival or growth and pluripotency. The NM23 tetramer or hexamer does not bind to the PSMGFR domain of the MUC1 * receptor and has the opposite function as that of a dimer. The hexamer NM23 induces stem cell differentiation.

Similarly, many researchers have attempted to develop drugs that target MUC1. However, we see that it is a cleavage type, called MUC1 *, which functions as a growth factor receptor, is activated by ligand-induced dimerization, and has an extracellular domain consisting primarily of PSMGFR sequences. Until the inventor discovered, it was unknown how MUC1 was associated with cancer. In fact, essentially all other attempts to develop MUC1 targeted anti-cancer therapies have targeted the longitudinal repetition of extracellular domains shed and released from the cell surface as found by the present inventor. ..
Until that time, the general wisdom was that MUC1 was cleaved, but the cleaved site, including tandem repetition, came to the transmembrane fragment that remained attached to the cell surface and bound to form a heterodimer. (Ligtenberg et al, 1990, Baruch A et al. (1999). As a double-staining experiment for cancer tissue, we are not true, but an antibody or missing domain that recognizes only a cleavage type (MUC1 *). By using an antibody that binds only to the (column repeat or core), it was revealed that the antibody that stains the cleavage type does not coexist (colocalize) with the antibody that binds to the column repeat. In fact, cancer. Most membrane staining of tissues was negative or minimally positive for MUC1 with a complete longitudinal fissure domain, but highly positive for truncated (clipped) MUC1 * types. Experiments have shown that large volumes of extracellular domains are released from the cell surface when MUC1 is cleaved (Mahanta, et al, 2008).

In addition to anti-cancer drugs, there have been many failures in attempts to develop cancer therapeutic vaccines. The problem is that the body's immune system creates antibodies against the "self" that destroy the target in healthy tissue as well as any future cancerous tissue. Several attempts have been made to develop cancer therapeutic vaccines that target MUC1. However, in each failed attempt, the portion of the targeted MUC1 molecule was the "core" previously shown by the inventor, known as the columnar repeating domain that sheds from the surface of cancer cells (Kroemer). G et al, 2013).

In one embodiment, the invention directs antibodies that preferentially recognize cancer cells rather than normal cells in which MUC1 is clipped to growth factor receptor morphology.

In another aspect, the invention is directed to an antibody that targets the NME protein.

In another aspect of the invention, the antibodies of the invention target NME proteins that are expressed preferentially in early life and to a lesser extent in adulthood. Preferably, these NME proteins are present at high levels in stem cells rather than in adult cells.

In another aspect of the invention, the antibodies of the invention are not expressed or have low levels of expression in adult tissues, but are preferentially expressed in the very early stages of embryogenesis, or in naive stem cells. Target NME proteins.

In another aspect of the invention, an antibody that targets NME1 is produced.

In another aspect of the invention, an antibody that targets NME6 is produced.

In another aspect of the invention, they produce antibodies that target NME1 or NME6, which inhibit dimerization.

In another aspect of the invention, an antibody that targets NME7 is produced.

In one embodiment of the invention, the antibody produced recognizes the NME protein and inhibits its dimerization. In another aspect of the invention, the antibody produced recognizes the NME protein and inhibits its interaction with MUC1. Also in another aspect of the invention, the antibody produced recognizes the NME protein and inhibits its interaction with MUC1 * or its interaction with the PSMGFR peptide.

Yet another aspect of the invention is the production of an antibody that binds to MUC1 * and inhibits its interaction with the NME protein. In one embodiment, they inhibit the interaction between MUC1 * and NME7 rather than between MUC1 * and NME1.

In one aspect of the invention, the antibody is produced outside the patient, eg, in cells, in an animal, or artificially, including using phage display and binding analysis. In another aspect of the invention, the antibody is produced in a patient and the portion of the protein targeted herein is administered alone or in combination, where an adjuvant can be added for use as a vaccine.

In one aspect, the invention is directed to an agent that inhibits the function of NME family proteins. The agent can be Fab, monovalent, divalent, or an antibody such as IgM, bispecific, human, or humanized. Alternatively, the drug can be a small molecule.
In one embodiment, the function of the NME family protein that can be sought for the inhibition can be the ability of the NME family protein to: promote stem cell proliferation and / or inhibit differentiation; promote cancer cell proliferation: And / or inhibit differentiation; bind to MUC1 *; bind to DNA; act as a transcriptional regulator; secreted by cells; or form a dimer. Specifically, the NME family member can preferably be NME7 or NME7-AB.

The agent can be an antibody that inhibits the tumorigenic activity of NME7 or NME7AB. Preferably, the NME family member can be a variant of NME7 with a molecular weight of 25-33 kDa. Alternatively, the NME family member can be NME6 or NME1.

In another aspect, the invention directs a therapeutic method comprising administering to a cancer patient or a patient at risk of developing cancer an effective amount of an agent that inhibits the tumorigenic activity of NME family proteins. The NME family protein can preferably be NME7, NME6 or NME1. In one embodiment, the agent can inhibit NME7 activity rather than NME1 activity. In another embodiment, the agent can block the binding between NME7 and MUC1 *. Alternatively, the drug may block the binding between NME7 and its cognate nucleic acid binding site. In yet another embodiment, the agent can be an antibody.

In another aspect, the invention directs a therapeutic method comprising administering to a cancer patient or a patient at risk of developing cancer an effective amount of NME1 as a hex summer. The NME1 polypeptide can be a hexamer-oriented mutant or variant.

In yet another aspect, the invention directs a therapeutic method comprising administering to a cancer patient or a patient at risk of developing cancer an effective amount of NME6 as a monomer. In one embodiment, NME6 can be a monomeric state oriented mutant or variant.

In yet another aspect, the present invention directs a therapeutic method comprising administering to a cancer patient or a patient at risk of developing cancer an effective amount of NME1 as a monomer. NME1 can be a monomeric state-oriented mutant or mutant.

In another embodiment, the invention administers an effective amount of a peptide or peptide mimetic that inhibits the interaction of its peer receptors with NME family members to a cancer patient or a patient at risk of developing cancer. Aim for treatment methods including. In one embodiment, the receptor of the same strain can be MUC1. In another embodiment, the peptide may be derived from the MUC1 * portion of MUC1, PSMGFR, N-10 PSMGFR, N-15 PSMGFR or N-20 PSMGFR.

In another aspect, the invention directs a method of classifying cancer or stratifying patients who carry or suspect cancer, including the following steps:
(i) Analyzing patient samples for the presence of stem or ancestral cell genes or gene products;
(ii) Then group patients who share similar expression or expression levels of the stem or ancestral cell gene or gene product.

(iii) The methods of the invention may further comprise the step (iii) of treating the patient with an agent that inhibits their stem or ancestral cell gene or gene product. Alternatively, the method may include the step (iii) of analyzing the stem or ancestral gene or gene product to assess the severity of the cancer, where the gene or gene product with the characteristics of the early stem or ancestral state. , Expression or higher expression indicates more active cancer, and expression or higher expression of a gene or gene product that is characteristic of the late ancestral state indicates less active cancer;
(iv) Design a treatment commensurate with the treatment of patients with more active or less active cancer as determined in step (iii);
(v) Treat the patient with treatment according to the design in step (iv). In such a method, the patient sample can be blood, body fluid or biopsy. Also, the gene or gene product can be an NME family protein. In one embodiment, the gene or gene product that exhibits early stem cell status can be NME7 or NME6.

In another aspect, the invention is directed to agents whose extracellular domain inhibits the interaction of NME family proteins and MUC1 transmembrane proteins lacking a columnar repeating domain, wherein the extracellular domain is: The drug binds to MUC1 * on cancer cells with a higher affinity than its binding to a MUC1 transmembrane protein that lacks the tandem repeat domain present in normal adult cells. In one embodiment, the agent comprises, without limitation, an antibody, a natural product, a synthetic chemical or a nucleic acid. In one embodiment, the NME family protein can be NME7, NME6 or bacterial NME.

In another embodiment, the present invention relates the extracellular domain to a MUC1 * on a cancer cell with a higher affinity for its binding to a MUC1 transmembrane protein lacking a columnar repeating domain on adult normal cells. Oriented to methods in which the extracellular domain inhibits the interaction between NME family proteins and MUC1 transmembrane proteins lacking a columnar repeating domain in the cell, including contacting the binding agent with the cell. In one embodiment, the agent may include, without limitation, antibodies, natural products, synthetic chemicals or nucleic acids. In one embodiment, the NME family protein can be NME7, NME6 or bacterial NME.

In another embodiment, the invention directs a method of identifying an agent that inhibits the interaction of an NME family protein and its extracellular domain lacking a columnar repeat domain MUC1 transmembrane protein, the step of which is cancer. Determining drug affinity for MUC1 * present on cells, determining drug affinity for MUC1 * present on stem or ancestral cells, and to MUC1 * present on stem or ancestral cells The ability to bind may include selecting a drug that binds by the MUC1 * present on the cancer cell, and thus identifying the drug. In one embodiment, the agent may include antibodies, natural products, synthetic chemicals or nucleic acids without limitation. In another embodiment, the stem or progenitor cell can be an embryonic stem cell, iPS cell, umbilical cord blood cell, bone marrow cell or hematopoietic progenitor cell. In one embodiment, the NME family protein can be NME7, NME6 or bacterial NME.

In another embodiment, the invention is directed to a recombinant mammal expressing a human NME protein in a germ cell and somatic cell, wherein the germ cell and somatic cell encode a human NME introduced into the mammal. Contains nucleic acids. Therefore, human NME can be expressed recombinantly in transgenic mammals. Of course, genetically modified mammals are not humans. In transgenic mammals, the NME protein can preferably be inducibly expressed. The NME protein can preferably be NME7 or NME7-AB.

In yet another embodiment, the invention comprises a method of producing a mammal that responds to cancer in a manner very similar to the human response, which is a mammal in which the human NME protein is expressed. Aim. The cancer may be spontaneously produced or implanted from cultured cells or humans. In one embodiment, the NME protein can be an NME1 dimer or an NME7 monomer. In another aspect, the mammal is a genetically modified mammal, the mammal containing germ cells and somatic cells containing recombinant human MUC1 or MUC1 * or NME protein gene sequences introduced into the mammal. Human MUC1 or MUC1 * or NME proteins can be expressed in cells and somatic cells. Preferably, the NME protein is inducibly expressed. Further, preferably, the NME protein can be NME7 or NME7-AB.

In another embodiment, the invention directs a method of increasing human tumor engraftment in a mammal, which involves introducing human tumor cells into a test mammal before introducing the cells into an NME1 dimer or NME7 monomer. Including mixing with.

In yet another embodiment, the invention is directed to a method of producing an antibody, which is to inject a mammal with an NME family protein or peptide fragment or fragment thereof, and to recover an antibody or antibody-producing cell. including. Preferably, the NME family protein can be NME7 or NME7-AB or NME1. Preferably, the peptide fragment can be selected from SEQ ID NOS: 88-140, preferably from 88-133, more preferably from 88-121.

In another aspect, the invention directs a method of producing an antibody or antibody-like molecule that specifically binds to an NME family protein or peptide fragment thereof, comprising the following steps:
(i) Screening for antibody or antibody fragment libraries or epitopes on NME family protein or peptide fragments;
(ii) Analyzing the binding of NME family proteins or their peptide fragments;
(iii) To identify specifically bound antibodies or antibody-like molecules.
The method comprises using a method known in the art to produce the identified antibody or antibody-like molecule for administration to a patient for the treatment or prevention of cancer. The NME family protein can be NME7 or NME7-AB or NME1. Preferably, the peptide fragment can be selected from SEQ ID NOS: 88-140, preferably 88-133, more preferably 88-121.

In yet another aspect, the invention directs a method of preventing cancer by vaccinating humans with NME family proteins or peptide fragments or fragments thereof. In one embodiment, the peptide fragment or multiple fragments comprises one or more peptides whose sequence is present in NME family proteins, which can optionally be mixed with a carrier or adjuvant, or attached to an immunogenic agent. NME family proteins can be NME1, NME6, NME7 or NME7-AB. Preferably, the peptide fragment can be selected from SEQ ID NOS: 88-140, preferably 88-133, more preferably 88-121. In a preferred embodiment, the peptide sequence is not a fragment of the human NME-H1 protein. The present invention is fully understood from the following detailed description and drawings, which are not limiting the invention.

Figure 1 is a graph of cancer cell growth analyzed as a function of divalent or monovalent antibody concentration, which shows that it is the dimerization of MUC1 * receptors that promotes growth. The growth of MUC1-positive breast cancer cells, ZR-75-30, was stimulated by the addition of divalent (Ab) anti-MUC1 * and inhibited by the addition of monovalent Fab. The addition of the divalent antibody resulted in a specific bell shape growth curve indicating growth factor receptor dimerization. Growth of MUC1-negative HEK 293 cells was unaffected by divalent or monovalent Fab anti-MUC1 *. When a divalent antibody is overloaded, there is one divalent antibody that binds to each receptor rather than one that dimerizes all two receptors, and results in growth. Inhibits. The vertical axis is normalized% cell growth, and the horizontal axis is antibody concentration. FIG. 2 is a graph of tumor volume analysis of T47D breast cancer cells engrafted in nu / nu female mice after treatment with vehicle or Fab of MN-E6 anti-MUC1 * antibody. Its efficacy of anti-MUC1 * E6 antibody was found to be statistically significant in reducing tumor volume with a p-value of 0.0001. Blocking the interaction of anti-MUC1 * Fab and MUC1 * NME inhibits cancer growth. The vertical axis is% normalized cell growth, and the horizontal axis is the day after the start of treatment. .. Figure 3A-3D. Photographs of Western blot gels showing expression of NME1 or NME7 in cell lysates of: 1) MUC1 * antibody surface (MN-C3 mab) coated surface with NM23-H1 dimer Cultured BGO1V human embryonic stem cells; 2) BGO1V human embryonic stem cells cultured by standard protocol with bFGF on layers of mouse feeder cells (MEF); 3) T47D cultured by standard method in RPMI medium Breast cancer cells; and 4) recombinant human NM23-H1 wilderness, "wt" (A, B). The lower row (C, D) shows the results of pull-down or immunoprecipitation analysis, where cell lysates were separately incubated in beds supplemented with MUC1 antibody to the cytoplasmic tail, "Ab-5". Species captured by binding to the MUC1 * peptide were separated by SDS-PAGE and blotted with antibodies against each individual NM23 protein. The same experiment was performed on NME6, but no data are shown. All NMEs have an NDPK domain, but enzymatic function does not require its role in pluripotency. NME7 has 2 NDPK domains and is a "natural" dimer. NME7 is expressed at higher levels in more naive stem cells. Pull-down analysis shows binding to MUC1 *. Figure 4A-4E shows Western blots in which cell lysates from T47D breast cancer cells, human ES (ES) cells of BGO1V and HES-3 and human SC101-A1 iPS cells were tested for the presence of NME1, NME6 or NME7. Shows a picture of. In all cell lines, NME1 had a clear molecular weight of ~ 17 kDa (A). For all cell lines, NME7-33 kDa species and 42 kDa species (C, E) could be detected in all cells except HES-3 cell line (cultured with FGF). Species that reacted with the NME6-specific antibody were detected in all cell lines except the HES-3 cell line when visualization was enhanced using sensitive signals. FIG. 5A-5C shows photographs of Western blots of human embryonic stem (ES) cells (A) and induced differentiated pluripotent stem (iPS) cells (B, C) tested for the presence of NME7. Western indicates the presence of three types of NME7 in cell lysates. Those with a clear molecular weight of ~ 42 kDa (overall), ~ 33 kDa (NME7-AB domain lacking N-terminal DH domain) and small ~ 25 kDa species. However, only lower molecular weight species are present in the conditioned medium (B). Figure 6A-6C. (A) is the elution profile of NME7-AB molecular sieving chromatography purification; (B) is a non-reducing SDS-PAGE gel from the NME7-AB peak fraction; (C) is the purified NME7 -A dissolution profile of AB molecular sieving chromatography. The recombinant NME7 novel variant "NME7-AB" containing NDPK domains A and B is well expressed in E. coli as its soluble protein at high production rates. Figure 7A-7C shows a photograph of nanoparticle binding analysis, where the MUC1 * extracellular domain peptide was immobilized on SAM-coated nanoparticles and the NME protein was added free to solution. The color change from pink to blue indicates that the free protein in solution can bind to two peptides simultaneously on two different nanoparticles. Figure 8 shows a graph of HRP signals from ELISA sandwich analysis, which shows that NME7-AB dimerizes MUC1 * extracellular domain peptides. The MUC1 * extracellular domain peptide immobilized on the plate was saturated by NME7. A second MUC1 * peptide with a C-terminal His-tag or biotin tag was added and visualized by an HRP-labeled antibody to His-tag or HRP-labeled streptavidin. FIG. 9A-9D shows an enlarged photograph of human iPS stem cells cultured in either recombinant NME7-AB or recombinant NM23 (NME1) purified dimer 1 day after planting. FIG. 10A-10D shows an enlarged photograph of human iPS stem cells cultured in either recombinant NME7-AB or recombinant NM23 (NME1) purified dimer 3 days after planting. Figures 11A-11D show photographs of immunocytochemistry experiments, showing that human HES-3 stem cells cultured in NME7-AB for teenage or higher passages are pluripotent markers NANOG (A), OCT3 / 4 (B). ), Tra 1-81 (C) and SSEA4 (D). Figure 12A-12C is a photograph of HES-3 embryonic stem cells stained with an antibody that recognizes trimethylated lysine 27 on histone 3, which shows that the cells are in a naive to primitive state [X chromosome, both Xs. (XaXi)], which is opposed to the activity of (XaXa), forms condensed dots. A) Cells were first cultured in FGF medium on MEF feeder cells (XaXi), B) then grown in NME7 during 10 passages (XaXa), C) then during 4 passages (XaXi). It was returned to FGF-MEF. Figure 13A-13G shows photographs of MUC1 * -positive cancer cells treated with none (row A), taxol (row B) or anti-NME7 antibody (row CE); (F) respond to treatment at 48 hours. A graph showing the number of cells and (G) dot-blotting used to estimate the antibody concentration used in the cancer cell inhibition experiment. Figure 14A-14K shows the results at 48 hours of experiment using anti-NME7 antibody to inhibit cancer cell growth. Photographs of cells cultured in anti-NME7 in medium at the concentrations shown in medium alone (A)), taxol (B) or (CJ); graph of cell number obtained using calcein. Is shown (K). Figure 15A-15K shows the results at 96 hours of experiment using anti-NME7 antibody to inhibit cancer cell growth. Photographs of cells cultured in anti-NME7 in medium at the concentrations shown in medium alone (A), taxol (B) or (CJ); graphs of cell numbers obtained using calcein Shown (K). Graphs and photographs show that anti-NME7 antibodies inhibit cancer cell growth at low concentrations in the nanomolar range. FIG. 16 shows a natural non-denatured gel showing the multimerized state of NM23-WT for three different preparations of recombinant NM23-S120G. Figure 17 A-17G. (A) shows a photograph of a non-reducing gel of NM23-WT, NM23-S120G-mixed, NM23-S120G dimer, which shows the multimerized state of three different preparations of wild-type protein and S120G mutant. Is shown. FIG. 17B shows the surface plasmon resonance (SPR) measurements of different NM23 multimers that bind to the MUC1 * extracellular domain peptide (PSMGFR) attached to the surface of the SPR chip. Figure 17C shows a photograph of a nanoparticle experiment showing that only the NM23 dimer binds to the receptor MUC1 * of the same strain. The MUC1 * extracellular domain peptide was immobilized on gold nanoparticles. Figure 17 D-G shows different NM23-H1 multimers tested for their ability to support pluripotent stem cell growth. FIG. 18 is a cartoon of the expression levels of NME7, NME1 dimer and NME1 hexmer based on the timing of expression and the analysis of the experimental examples of the present application. Figure 19A-19I shows a graph of the results of the ELISA analysis, showing that human NME6 binds to the PSMGFR peptide in the MUC1 * extracellular domain. Recombinant NME6-wt was separated into monomers or multimers by FPLC and analyzed by ELISA for its ability to bind to the surface of PSMGFR peptides (A). NME6 multimers were dissociated by dilution with SDS by the CMC (critical micelle concentration) fraction and then analyzed by ELISA for their ability to bind to the surface of the PSMGFR peptide (B). The NME6 mutant designed to be dimerized is generated by mimicking the dimer formation oriented NME1 S120G mutant and is S139G in NME6 by alignment. The second mutant is made by displacing residues in its critical area where human NME6 is to be converted, similar to sponge NME6, which has been reported to exist as a dimer. These recombinant mutants were expressed, purified and analyzed for their ability to bind PSMGFR peptides to the surface (C). DI is a photograph of a polyacrylamide gel demonstrating the expression of various recombinant human NME6 proteins. D) NME6 wt is expressed. E) NME6, which carries the S139G displacement, which corresponds to the S120G displacement in NME1, was expressed. F) Human NME6 carrying displacements S139A, V142D and V143A that mimic sponge NME6 reported to be a dimer. G, H) (GSSS) single chain human with two domains joined by _three linkers NME6. I) Pull-down analysis was performed using an antibody against the C-terminus of MUC1. The protein bound to MUC1 was dissociated on the gel and then tested with an antibody against NME6. The gel expressed NME6 bound to MUC1 in T47D breast cancer cells, BGo1v and HES-3 human embryonic stem cells, and human iPS cells. FIG. 20A-20D shows Western blot photographs, where cell lysates (A, C) or nuclear fractions from T47D breast cancer cells, BGO1V, and HES-3 human ES cells and human SC101-A1 iPS cells. (B, D) was tested for the presence of NME7 (A, B) or NME1 (C, D). FIG. 21 is a graph of real-time PCR analysis of NME1, NME6, NME7 and MUC1 for MUC1-positive T47D breast cancer cells, MUC1-positive DU145 prostate cancer cells and MUC1-negative PC3 prostate cancer cells. Analysis is associated with 18S ribosome RNA and standardized for analysis of T47D cells. Both MUC1-positive cancer cell lines are high in NME7. MUC1-negative cell lines lacked any detectable NME1, NME7 or MUC1 but had very high expression of NME6. Figure 22 shows a photograph of Western blot, where stem cell lysates (odd numbered lanes) or cell-conditioned medium (even numbered lanes) were tested for the presence of NME7. iPS (induced differentiated pluripotent stem) cells are FGF (lanes 1 and 2) on MEF, NM23-H1 dimer (lanes 3 and 4) on the surface of anti-MUC1 * antibody (C3), or anti-MUC1. * Cultured in NME7 (lanes 5-8) on the surface of antibody (C3). HES-3 (human embryo stem) cells are FGF (lanes 9 and 10) on MEF, NM23-H1 dimer (lanes 11 and 12) on the surface of anti-MUC1 * antibody (C3), or anti. MUC1 * antibody (C3) was cultured on the surface with NME7 (lanes 13-14). Mouse embryo fibroblast (MEF) cells were also examined (lanes 15 and 16). Western blots show that the cell lysate contains 7 NME species with a molecular weight of ~ 42 kDa, which corresponds to a full-length protein. However, the secreted species develops with a clear molecular weight of ~ 33 kDa, which corresponds to the NME7 species lacking the N-terminal leader sequence. Figures 23A-23B are photographs of the same Western blot shown in Figure 22 to identify recombinant NM23-H1, ~ 17 kDa and NME7-AB 33 kDa (stem cells were cultured in lanes 3-8 and 11-14). , Striped and tested for the presence of histidine-tagged species. The results of minimal staining showed that the major NME7 species detected in FIG. 22 were native NME7 produced and processed by stem cells. Figure 24A-24B shows various cell lysates and corresponding preparations tested for the presence of NME7 using a mouse monoclonal antibody (A) or another monoclonal antibody (B) that recognizes only the N-terminal DM10 sequence. A photograph of a Western blot of the finished medium is shown. Lack of DM10-specific antibody binding to ~ 33 kDa NME7 species in samples from cell-prepared medium is that the secreted form of NME7 lacks most, if not all, of the N-terminal DM10 leader sequence. Indicates that you are. Figure 25A-25B. (A) shows a polyacrylamide gel of NME from the bacterium Halomonas Sp593, which was expressed in E. coli and expressed as a soluble protein and a natural dimer. (B) shows that NME from Halomonas Sp593 binds to the PSMGFR peptide in the MUC1 * extracellular domain in ELISA analysis. Figure 26 shows a polyacrylamide gel of NME from the bacterium Porphyromonas gingivalis W83. Figure 27A-27C. (A) shows the sequence alignment of Halomonas Sp 593 bacterial NME to human NME-H1. (B) shows the sequence alignment of Halomonas Sp 593 bacterial NME to the human NME7-A domain. (C) shows the sequence alignment of Halomonas Sp 953 bacterial NME to the human NME7-B domain. Figures 28A-28D are photographs of human embryonic stem cells cultured in bacterial NME from Halomonas Sp 593 at 10X magnification (A, C) or 20X magnification (B, D). Figure 29 shows OCT4 stem / cancer cell markers after human fibroblasts were cultured in serum-free medium containing either human NME7-AB, human NME1 dimer or bacterial NME from Halomonas Sp 593. It is a graph of RT-PCR data for analyzing the expression of. Figure 30 shows RT-PCR analysis of expression levels of the stem / oncogenes OCT4 and NANOG in fibroblasts cultured in the presence of human NME7-AB, human NME1 or Halomonas Sp 593-derived bacterial NME "HSP 593". It is a graph of. In some cases, the low kinase inhibitor "ROCi" was added to attach to the surface of non-adherent cells (stem / cancerous). FIG. 31 is a photograph showing human fibroblasts after 8 days in culture in serum-free medium containing dimeric form of human NME1 at 4X magnification. FIG. 32 is a photograph showing human fibroblasts after 18 days in culture in serum-free medium containing dimeric form of human NME1 at 20X magnification. FIG. 33 is a photograph showing human fibroblasts after 18 days in culture in serum-free medium containing bacterial NME from Halomonas Sp 593 at 4X magnification. FIG. 34 is a photograph showing human fibroblasts after 18 days in culture in serum-free medium containing bacterial NME from Halomonas Sp 593 at 20X magnification. Figure 35 shows photographs of human fibroblasts after 18 days in culture in serum-free medium containing human NME7-AB at 4X magnification. Figure 36 shows photographs of human fibroblasts after 18 days in culture in serum-free medium containing human NME7-AB at 20X magnification. Figure 37 shows photographs of human fibroblasts after 18 days in standard medium without NME protein at 4X magnification. Figure 38 shows photographs of human fibroblasts after 18 days in standard medium without NME protein at 20X magnification. FIG. 39 is a graph of RT-PCR analysis of expression levels of transcription factors BRD4 and cofactor JMJD6 in the earliest stages of naive human stem cells compared to late stage primed stem cells. Figure 40 shows the expression levels of chromatin rearrangement factors that are suppressed when fibroblasts return to a state of inducible differentiation pluripotency while other factors are suppressed in naive stem cells and some cancer cells. It is a graph of RT-PCR analysis. Expression levels of the chromatin rearrangement genes Brd4, JMJD6, Mbd3 and CHD4 were measured in fibroblasts cultured in the presence of bacterial NME from human NME7-AB, human NME1 or Halomonas Sp 593, "HSP 593". .. In some cases, the low kinase inhibitor "ROCi" was added to attach to the surface of non-adherent cells (stem / cancerous). Figure 41 shows a mixture of RT-PCR analysis of stem / oncogene expression levels in fibroblasts cultured in the presence of bacterial NME from human NME7-AB, human NME1 or Halomonas Sp 593, "HSP 593". It is a graph. In some cases, the raw kinase inhibitor "ROCi" was added to attach to the surface of non-adherent cells (stem / cancerous). Figure 42 is cultured in the presence of bacterial NME from human NME7-AB, human NME1 or Halomonas Sp 593 "HSP 593" using a compressed Y-axis to better show the differences in genes with smaller displacements. It is a mixed graph of RT-PCR analysis of the expression level of stem / oncogene in fibroblasts. In some cases, the low kinase inhibitor "ROCi" was added to attach to the surface of non-adherent cells (stem / cancerous). FIG. 43 is a graph of RT-PCR analysis of stem cell markers and gene expression of cancer stem cell markers for T47D cancer cells after culturing in a common medium or medium containing NME7, where non-adherent (floating). The cells that became (things) were analyzed separately from those that remained attached. FIG. 44 is a graph of RT-PCR analysis for gene expression of the cancer stem cell marker CXCR4 in stem cell markers SOX2 and T47D cancer cells. Cells were cultured in either conventional medium or medium containing NME1 dimer or NME7 (NME7-AB). Cell types analyzed separately were suspended cells, with all cells attached to the raw kinase inhibitor (+ Ri) and cells, or in the absence of the raw kinase inhibitor (-Ri). It is a cell that remains after attachment. FIG. 45 is a graph of RT-PCR analysis of gene expression of various stems of T47D breast cancer cells and putative cancer stem cell markers. Cells were cultured in either conventional medium or medium containing NME1 dimer (“NM23”) or NME7 (NME7-AB). Cell types analyzed separately were suspended cells, with all cells attached to the raw kinase inhibitor (+ Ri) and cells, or in the absence of the raw kinase inhibitor (-Ri). It is a cell that remains after attachment. FIG. 46 is a graph of RT-PCR analysis of gene expression of various stem and putative cancer stem cell markers for DU145 prostate cancer cells. Cells were cultured in either conventional medium or medium containing NME1 dimer (“NM23”) or NME7 (NME7-AB). By passage 2, the cells remained attached and no raw kinase inhibitor was used. FIG. 47 is a graph of RT-PCR analysis of gene expression of various stem and putative cancer stem cell markers for DU145 prostate cancer cells. Cells were cultured in either conventional medium or medium containing the NME1 dimer (“NM23”). By passage 2, the cells remained attached and no raw kinase inhibitor was used. Figure 48 shows T47D breast cancer from culture in minimal serum-free basal medium in which the only factor added was either a "2i" inhibitor (GSK3 beta and MEK inhibitor) or a human recombinant NME7-AB. RT-PCR analysis graphs of expression levels of the reported "cancer stem cell" or "tumor-inducing cell" markers CDH1 (E-cadherin), CXCR4, NANOG, OCT4 and SOX2 in addition to MUC1 in cells. The cells analyzed were anchorage-independently beginning to grow as suspended matter. Figure 49 shows T47D breast cancer from culture in minimal serum-free basal medium in which the only factor added was either a "2i" inhibitor (GSK3 beta and MEK inhibitor) or a human recombinant NME7-AB. Expression levels of transcription factors BRD4 and cofactor JMJD6, which were reported to suppress NME7 and induce NME1, respectively, and chromatin rearrangement factors MBD3 and CHD4, which were reported to block the induction of stem cell pluripotency, in cells. It is a graph of RT-PCR analysis. FIG. 50 is a diagram of the interaction map of related factors and NME7 by analysis of the experimental example of the present application. FIG. 51 is a graph of tumor volume analyzed over time. T47D breast cancer cells are transplanted using standard analysis (dotted line) or where the cells are mixed with NME7-AB at 50/50 volume / volume and after 10 days, those mice inject NME7-AB daily. Was done. Figure 52 shows MMP14, MMP16 and ADAM17 capable of degrading MUC1 full length into MUC1 * in cultured cancer cells (T47D) or human embryonic stem cells (HES) cultured in FGF, NM23-H1 dimer or NME7. The graph of the quantitative PCR analysis which analyzed the RNA expression of is shown. FIG. 53 shows FGF-grown HES-3 human embryonic stem cells, human NME7-AB-grown HES-3 cells, in vitro T47D breast cells, and animal-transplanted T47D breast cells. FIG. 5 is a graph of RT-PCR analysis of expression levels of cleavage enzymes MMP14, MMP16 and ADAM17 in DU145 prostate cancer cells in vitro, in DU145 cells transplanted into animals, and in 1500 breast cancer cells transplanted into animals. .. All cells were standardized to HES-3 cells grown in FGF at MEF. Figure 54 shows T47D breast cancer cells in vitro, HES-3 human embryonic stem cells grown in FGF, HES-3 cells grown in human NME7-AB, and HES-3 grown in NME1 dimer. Graphs of RT-PCR analysis of expression levels of cleavage enzymes MMP14, MMP16 and ADAM17 in cells, all cells standardized for T47D breast cancer cells in vitro. Figure 55 is a graph of tumor volume analysis of DU145 hormone refractory prostate cancer cells, showing results 60 days after transplantation into male mice with NOD / SCID and treatment with a vehicle or Fab of MN-E6 anti-MUC1 * antibody. From the 60th to the 70th, the treatment group was switched. The efficacy of the anti-MUC1 * E6 antibody was statistically significant in reducing tumor volume with a p-value of 0.0001. Figure 56 is a graph of RT-PCR analysis of expression levels of the cleavage enzymes MMP14 and MMP16 in tumors resected from DU145 hormone refractory prostate cancer cells, transplanted into NOD / SCID male mice and MN-E6 anti-MUC1. * Results of 60 days of treatment with antibody vehicle or Fab. Treatment to block the MUC1 * growth factor receptor reduced the expression of both cleaving enzymes, but only MMP14 was statistically significant. Figures 57A-57B. (A) is a photograph of Western blot examination of MUC1 * in a tumor resected from DU145 hormone refractory prostate cancer cells, transplanted into male mice with NOD / SCID, and in a vehicle or Fab of MN-E6 anti-MUC1 * antibody. Is the result of 60 days of treatment. The photographs show that anti-MUC1 * Fab-treated mice have less MUC1 *, i.e. less MUC1 cleavage in the treatment group. (B) is a graph of RT-PCR analysis of microRNA-145 expression levels in tumors resected from DU145 hormone refractory prostate cancer cells, transplanted into NOD / SCID male mice, and MN-E6 anti-MUC1 *. Results of 60 days of treatment with antibody vehicle or Fab. The graph shows that, on average, miR-145, which signals to differentiate into stem cells, is increased in the treatment group compared to the control group. Figures 58A-58D show the results of a FACS experiment in which live cancer cells were tested with a stem cell-specific anti-MUC1 * monoclonal antibody or a cancer cell-specific monoclonal antibody. (A) The MN-C2 monoclonal antibody selected based on the binding priority to the N-10 peptide shows strong binding to raw T47D breast cancer cells and is cancer cell specific. (B) The MN-C3 monoclonal antibody selected based on the binding priority to the C-10 peptide shows non-binding to raw T47D breast cancer cells and is stem cell specific. C) Cancer cell-specific MN-C2 binds to DU145 prostate cancer cells. D) Stem cell-specific MN-C3 does not bind to DU145 prostate cancer cells. E) Graphs from another FACS experiment show that cancer cell-specific monoclonal antibodies MN-C2 and MN-E6 bind to DU145 prostate cancer cells, while stem cell-specific MN-C3 does not. Figures 59A-59D show the results of FACS experiments in which stem cells or cancer cells were tested with stem cell-specific anti-MUC1 * monoclonal antibody or cancer cell-specific monoclonal antibody. Stem cell-specific MN-C3 clones showed strong binding to BGO1v human embryonic stem cells (A), but not to T47D breast cancer cells (B) or DU145 prostate cancer cells (C). A graph from another FACS experiment shows that stem cell-specific MN-C3 monoclonals show strong binding to stem cells, but T47D breast cancer cells, 1500 breast cancer cell lines, DU145 prostate cancer cells or MUC1-negative PC3 prostate cancer cells (D). Does not combine with. Figure 60A-60E shows photographs of DU145 prostate cancer cells cultured in normal medium with the following additions; none (A), stem cell-specific MN-C3 Fab (B), stem cell-specific MN- Fab (C) of C8, Fab (D) of cancer cell-specific MN-C2 or Fab (E) of cancer cell-specific MN-E6. If the antibody recognized MUC1 * as it appeared in cancer cells, the antibody Fab would have blocked MUC1 * dimerization and induced cell death. As can be seen in the photograph, the stem cell-specific antibody Fabs had no effect on cancer cell growth, while the cancer cell-specific antibody Fabs effectively killed cancer cells. FIG. 61 is a sequence alignment between human NME1 and human NME7-A or -B domains. FIG. 62 lists immunogenic peptides from human NME7 with low sequence identity to NME1. The peptide sequences listed are identified as immunogenic peptides that give rise to antibodies that target human NME7 rather than human NME1. The sequence was chosen due to lack of sequence homology to human NME1 and is useful as an NME7-specific peptide for producing antibodies that inhibit NME7 for the treatment or prevention of cancer. Figure 63 lists immunogenic peptides from human NME7 that may be important for structural integrity or for binding to MUC1 *. Bivalent and bispecific antibodies, in which each variable domain binds to different peptide moieties of NME7, are preferred. Such peptides can be produced by using one or more peptides to produce antibodies specific for both. This peptide is useful as an NME7-specific peptide for producing antibodies that inhibit NME7 for the treatment or prevention of cancer. Figure 64 lists immunogenic peptides from human NME1 that may be important for structural integrity or for binding to MUC1 *. The listed peptide sequences were selected from human NME1 by their high homology to human NME7 as well as their homology to other bacterial NME proteins capable of mimicking their function. Peptides 50-53 have high homology, especially to human NME7-A or -B, and even HSP 593.

Definition

The MUC1 * extracellular domain used here is primarily the PSMGFR sequence:
Defined by (GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQID NO: 6)). The exact site of MUC1 cleavage depends on the enzyme that cuts it, and the cleavage enzyme mutates depending on the cell type, tissue type, or time in cell evolution / development, so the exact sequence of the MUC1 * extracellular domain. May mutate at the N-terminal.

As used herein, the term "PSMGFR" is an acronym for the primary sequence of the MUC1 growth factor receptor, as described as GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 6). In this regard, an "N-number" such as in "N-10 PSMGFR", "N-15 PSMGFR" or "N-20 PSMGFR" refers to the number of amino acid residues deleted at the N-terminus of PSMGFR. Point to. Similarly, a "C-number" such as in "C-10 PSMGFR", "C-15 PSMGFR" or "C-20 PSMGFR" refers to the number of amino acid residues deleted at the C-terminus of PSMGFR.

As used herein, the extracellular domain of MUC1 * refers to the extracellular portion of the MUC1 protein that lacks the column repeat domain. Often, MUC1 * is a degradation product consisting of a short extracellular domain in which the MUC1 * moiety is column-repeated, lacking a transmembrane domain and cytoplasmic tail. The exact location of MUC1 cleavage is unknown, as it appears to be cleaved by one or more enzymes. The MUC1 * extracellular domain contains most of the PSMGFR sequence, but may have additional 10-20 N-terminal amino acids.

The numbered 1-10 of "NME family proteins" or "NME family proteins" used herein were all grouped by having at least one NDPK (nucleotide diphosphate kinase) domain. It is a protein. In some cases, the NDPK domain is not functional in the sense that it can catalyze the conversion of ATP to ADP. The NME protein was officially known as the NM23 protein, numbered H1, H2, etc. Here, the terms NM23 and NME are compatible. Here, the terms NME1, NME2, NME6 and NME7 are used to refer to both NME variants as well as natural proteins. In some cases, these variants are more soluble, better expressed in E. coli, or more soluble than natural sequences / proteins. For example, NME7 as used herein has excellent commercial applicability because the mutation allows solubilization in E. coli and high production expression of properly folded proteins. May mean a natural protein or variant, such as. As quoted here, "NME1" is compatible with "NM23-H1". It is further intended that the present invention is not limited by the exact sequence of the NME protein. The mutant NME1-S120G, called NM23-S120G, is used interchangeably throughout the specification. S120G and P96S mutants are preferred here because of their suitability for dimer formation, but are referred to here as NM23 or NME1 dimers.

The NME7 cited here is a natural NME7 with a molecular weight of 42 kDa, a cleaved type with a molecular weight between 25 and 33 kDa, a mutant lacking a DM10 leader sequence, NME7-AB, or a recombinant NME7 protein, or its sequence is efficient. It is intended to mean these variants that are expressive, increase in high molecular weight, soluble or changeable to other properties that make NME7 more effective or more commercially available.

The present invention discloses antibodies and antibody variants that regulate pathways for MUC1 *, where a set of antibodies preferentially resides in stem cells but does not sufficiently recognize MUC1 * on cancer cells. , Combine with MUC1 *. Another set of antibodies preferentially binds to MUC1 *, which is present in cancer cells but does not fully recognize MUC1 * on stem cells. The present invention further demonstrates methods for identifying other antibodies that fall into these categories. The present invention, hereinafter referred to as "stem cell antibody", further illustrates a method of using a first set of antibodies for stimulating stem cell growth in vitro and in vivo. The present invention is further referred to as "cancer cell antibody" and describes a method of using a second set of antibodies to inhibit cancer cell growth in vitro and in vivo.

In the description of the present specification, the cancer-specific antibody MIN-C2 (in addition, cited herein as in the application in which the present application claims priority as "C2") or MIN-E6 (in addition, the present application is "E6"). (Cited here as well as the application claiming priority as) is the same structurally and sequencely antibody cited in this application by the applicant as in other applications. A description of these antibodies and their CDR sequences can be found in WO2010 / 042562 (PCT / US2009 / 059754) filed October 6, 2009. In particular, see FIGS. 11-16 there.

Similarly, stem cell-specific antibody 2D6C3 (in addition cited herein as in the application claiming priority as "C3") or MN-C3 or 2D6C8 (plus this application claiming priority as "C8"). (Cited herein as in the application) or MN-C8 is the structurally and sequencely identical antibody cited in this application by the applicant as in other applications. Descriptions of these antibodies and their CDR sequences can be found in WO2012 / 126013 (PCT / US2012 / 059754) filed March 19, 2012. In particular, see FIGS. 13-18 there.

In the description of the present specification, "effective amount of a drug that inhibits an NME family protein" is used when interfering with an activation interaction between an NME family protein and a receptor of the same strain, such as MUC1 or MUC1 *. Refers to the effective amount of the drug in.

As used herein, "high homology" means at least 30%, 35%, 40%, 45%, 50%, 55%, 60% of the specified overlapping domains between any two polypeptides. , 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% are considered to have the same identity.

In the description herein, "low homology" means having less than 25%, 20%, 15%, 10% or less than 5% identity of the specified overlapping domain between any two polypeptides. Is considered to be.

For a drug referred to herein as a "small molecule," it has a molecular weight between 50 Da and 2000 Da, more preferably between 150 Da and 1000 Da, and even more preferably between 200 Da and 750 Da. It can be a synthetic chemical molecule with or a chemistry-based molecule.

As long as the molecule exists in nature in the description herein, it can be a chemical or biological molecule with respect to a drug called a "natural product".

In the description herein, "2i inhibitor" refers to a small molecule inhibitor of GSK3 beta and MEK of the MAP kinase signal pathway. The name 2i was created in the research literature (Silva J et al., 2008). However, here "2i" is either GSK3 beta or MEK so that if they inhibit the target, there are many small molecules or biological factors that have the same effect on pluripotency or tumorigenesis. Refers to any inhibitor of that.

In the description of the present application, FGF, FGF-2 or bFGF refers to fibroblast growth factor.

As described herein, a "Rho-related kinase inhibitor" can be a small molecule, peptide or protein (Rath N, et al., 2012). Raw kinase inhibitors are abbreviated here and either as ROCi, ROCKi or Ri. The use of specific raw kinase inhibitors is intended to be exemplary and can be used as an alternative to other raw kinase inhibitors.

As used herein, the term "cancer stem cell" or "tumor initiating cell" refers to a cancer cell that expresses a level of gene linked to a more metastatic state or a more active cancer. The term "cancer stem cell" or "tumor initiating cell" can further refer to a cancer cell that requires a much smaller number of cells to develop a tumor when transplanted into an animal. "Cancer stem cells" or "tumor-initiating cells" are often resistant to chemotherapeutic agents.

In the description of the present specification, the terms "stem / cancer", "cancer-like", and "stem-like" mean that a cell obtains the characteristics of a stem cell or a cancer cell, and the gene expression profile of the stem cell, the cancer cell, or the cancer stem cell is important. Means a state that shares various elements. Stem-like cells can be somatic cells that undergo induction into a less mature state, such as increased expression of pluripotent genes. Stem-like cells are further referred to cells that have undergone certain dedifferentiation or are in a meta-stable state in which they can alter their final differentiation. Cancer-like cells are cancer cells that are not fully characterized but exhibit the morphology and characteristics of cancer cells so that they can grow anchorage-independently or develop tumors in animals. It is possible.

In the description herein, the term "antibody-like" means a molecule that can be prepared such that it contains a portion of an antibody but is not a naturally occurring antibody in nature. Examples include, but are not limited to, CAR (chimeric antigen receptor) T cell technology and Ylanthia technology. CAR technology uses antibody epitopes fused to parts of T cells to direct the body's immune system to attack specific target proteins or cells. The Ylanthia technique consists of an "antibody-like" library, a collection of synthetic human Fabs that are then screened for binding to peptide epitopes from the target protein. The selected Fab regions are then prepared on the scaffold or framework so that they resemble antibodies.

Sequence listing free text

The use of nucleotide symbols other than a, g, c and t is subject to the agreement described in WIPO Standard ST.25, Appendix 2, Table 1. Where k stands for t or g; n stands for a, c, t or g; m stands for a or c; r stands for a or g; s stands for c or g; w stands for a or g And y represent c or t.

MTPGTQSPFF LLLLLTVLTV VTGSGHASST PGGEKETSATQRSSVPSSTE KNAVSMTSSV LSSHSPGSGS STTQGQDVTL APATEPASGS AATWGQDVTS VPVTRPALGSTTPPAHDVTS APDNKPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGSTAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGSTAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGSTAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGSTAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGSTAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGSTAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGSTAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGSTAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGSTAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDNRPALGS TAPPVHNVTS, ASGSASGSAS TLVHNGTSAR ATTTPASKST PFSIPSHHSD TPTTLASHST KTDASSTHHSSVPPLTSSNH STSPQLSTGV SFFFLSFHIS NLQFNSSLED PSTDYYQELQ RDISEMFLQI YKQGGFLGLSNIKFRPGSVV VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPFPFSA QSGAGVPGWGIALLVLVCVL VALAIVYLIA LAVCQCRRKN YGQLDIFPAR DTYHPMSEYP TYHTHGRYVP PSSTDRSPYEKVSAGNGGSS LSYTNPAVAA ASANL (SEQ ID NO: 1)
Described the full-length MUC1 receptor (mucin 1 precursor (Genbank registration number): P15941).

MTPGTQSPFFLLLLLTVLT (SEQ ID NO: 2)

MTPGTQSPFFLLLLLTVLT VVTA (SEQ ID NO: 3)

MTPGTQSPFFLLLLLTVLT VVTG (SEQ ID NO: 4)

SEQ ID NOS: 2, 3 and 4 identify N-terminal MUC-1 signaling sequences on the cell membrane surface that are oriented towards MUC1 receptors and shortened isoforms. Up to 3 amino acid residues are absent at the C-terminus as indicated by variants of SEQ ID NOS: 2, 3 and 4.

GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANL (SEQID NO: 5)
It has a nat-PSMGFR at its N-terminus, identifies a shortened MUC1 receptor, and contains a full-length MUC1 receptor transmembrane and cytoplasmic sequence.

GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQID NO: 6)
This identifies the natural primary sequence of the MUC1 growth factor receptor (nat-PSMGFR: an example of "PSMGFR").

TINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQID NO: 7)
This identifies the natural primary sequence of the MUC1 growth factor receptor (nat-PSMGFR: an example of "PSMGFR") with a single amino acid deletion at the N-terminus of SEQ ID NO: 6.

GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (SEQID NO: 8)
This identifies a functional variant of the natural primary sequence "SPY" of the MUC1 growth factor receptor with enhanced stability (var-PSMGFR: an example of "PSMGFR").

TINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (SEQID NO: 9)
It is a natural MUC1 growth factor receptor with enhanced stability (var-PSMGFR- "PSMGFR" example) with a single amino acid deletion at the C-terminus of SEQ ID NO: 8. Identify functional variants of the primary sequence "SPY".

tgtcagtgccgccgaaagaactacgggcagctggacatctttccagcccgggatacctaccatcctatgagcgagtaccccacctaccacacccatgggcgctatgtgccccctagcagtaccgatcgtagcccctatgagaaggtttctgcaggtaacggtggca
This identifies the nucleotide sequence of the MUC1 cytoplasmic domain.

CQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANL (SEQID NO: 11)
This identifies the amino acid sequence of the MUC1 cytoplasmic domain.

gagatcctgagacaatgaatcatagtgaaagattcgttttcattgcagagtggtatgatccaaatgcttcacttcttcgacgttatgagcttttattttacccaggggatggatctgttgaaatgcatgatgtaaagaatcatcgcacctttttaaagcggaccaaatatgataacctgcacttggaagatttatttataggcaacaaagtgaatgtcttttctcgacaactggtattaattgactatggggatcaatatacagctcgccagctgggcagtaggaaagaaaaaacgctagccctaattaaac, (SEQID NO: 12)
This identifies the NME7 nucleotide sequence (NME7: GENBANK ACCESSION AB209049).

DPETMNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGMLNTLYSVHFVNRRAMFIFLMYFMYRK (SEQID NO: 13)
This identifies the NME7 amino acid sequence (NME7: GENBANK ACCESSION AB209049).

(SEQID NO: 14)
This identifies the NM23-H1 nucleotide sequence (NM23-H1: GENBANK ACCESSION AF487339).

MVLLSTLGIVFQGEGPPISSCDTGTMANCERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVGLKFMQASEDLLKEHYVDLKDRPFFAGLVKYMHSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGSDSVESAEKEIGLWFHPEELVDY
This identifies the NM23-H1 amino acid sequence (NM23-H1: GENBANK ACCESSION AF487339).

(SEQID NO: 16)
This identifies the NM23-H1 S120G mutant nucleotide sequence (NM23-H1: GENBANK ACCESSION AF487339).

MVLLSTLGIVFQGEGPPISSCDTGTMANCERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVGLKFMQASEDLLKEHYVDLKDRPFFAGLVKYMHSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGGDSVESAEKEIGLWFHPEELVDY
This identifies the NM23-H1 S120G mutant amino acid sequence (NM23-H1: GENBANK ACCESSION AF487339).

atggccaacctggagcgcaccttcatcgccatcaagccggacggcgtgcagcgcggcctggtgggcgagatcatcaagcgcttcgagcagaagggattccgcctcgtggccatgaagttcctccgggcctctgaagaacacctgaagcagcactacattgacctgaaagaccgaccattcttccctgggctggtgaagtacatgaactcagggccggttgtggccatggtctgggaggggctgaacgtggtgaagacaggccgagtgatgcttggggagaccaatccagcagattcaaagccaggcaccattcgtggggacttctgcattcaggttggcaggaacatcattcatggcagtgattcagtaaaaagtgctgaaaaagaaatcagcctatggtttaagcctgaagaactggttgactacaagtcttgtgctcatgactgggtctatgaataa (SEQID NO: 18)
This identifies the NM23-H2 nucleotide sequence (NM23-H2: GENBANK ACCESSION AK313448).

MANLERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVAMKFLRASEEHLKQHYIDLKDRPFFPGLVKYMNSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGSDSVKSAEKEISLWFKPEELVDYKSCAHDWVYE (SEQID NO: 19)
This identifies the NM23-H2 amino acid sequence (NM23-H2: GENBANK ACCESSION AK313448).

Human NM23-H7-2 sequence optimized for E. coli expression:

(DNA)

atgcatgacgttaaaaatcaccgtacctttctgaaacgcacgaaatatgataatctgcatctggaagacctgtttattggcaacaaagtcaatgtgttctctcgtcagctggtgctgatcgattatggcgaccagtacaccgcgcgtcaactgggtagtcgcaaagaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatact ttttcaaaattctggataattga (SEQID NO: 20)

(amino acid)

MHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKT, KIQNAVHCTDLPEDGLLEVQYFFKILDN- (SEQ ID NO: 21)

Human NME7-A:

(DNA)

atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgtttttttga (SEQID NO: 22)

(amino acid)

MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFF-(SEQ ID NO: 23)

Human NME7-A1:

(DNA)

atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttacttga (SEQID NO: 24)

(amino acid)

MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFT-(SEQ ID NO: 25)

Human NME7-A2:

(DNA)

atgaatcatagtgaaagattcgttttcattgcagagtggtatgatccaaatgcttcacttcttcgacgttatgagcttttattttacccaggggatggatctgttgaaatgcatgatgtaaagaatcatcgcacctttttaaagcggaccaaatatgataacctgcacttggaagatttatttataggcaacaaagtgaatgtcttttctcgacaactggtattaattgactatggggatcaatatacagctcgccagctgggcagtaggaaagaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgtttttttga (SEQ ID NO: 26)

(amino acid)

MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDA

Human NME7-A3:

(DNA)

atgaatcatagtgaaagattcgttttcattgcagagtggtatgatccaaatgcttcacttcttcgacgttatgagcttttattttacccaggggatggatctgttgaaatgcatgatgtaaagaatcatcgcacctttttaaagcggaccaaatatgataacctgcacttggaagatttatttataggcaacaaagtgaatgtcttttctcgacaactggtattaattgactatggggatcaatatacagctcgccagctgggcagtaggaaagaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttacttga (SEQID NO: 28)

(amino acid)

MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRD

Human NME7-B:

(DNA)

atgaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttctga (SEQID NO: 30)

(amino acid)

MNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF-(SEQ ID NO: 31)

Human NME7-B1:

(DNA)

atgaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggataattagtga (SEQID NO: 32)

(amino acid)

MNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN-(SEQ ID NO: 33)

Human NME7-B2:

(DNA)

atgccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttctga (SEQID NO: 34)

(amino acid)

MPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF-(SEQ ID NO: 35)

Human NME7-B3:

(DNA)

atgccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggataattagtga (SEQID NO: 36)

(amino acid)

MPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN-(SEQ ID NO: 37)

Human NME7-AB:

(DNA)

atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggataattagtga (SEQID NO: 38)

(amino acid)

MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN - (SEQ ID NO: 39)

Human NME7-AB1:

(DNA)

atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatg, gtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttctga (SEQID NO: 40)

(amino acid)

MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF- (SEQ ID NO: 41)

Human NME7-A sequence optimized for E. coli expression:

(DNA)

atggaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttctga (SEQID NO: 42)

(amino acid)

MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFF-(SEQ ID NO: 43)

Human NME7-A1 sequence optimized for E. coli expression:

(DNA)

atggaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttacctga (SEQID NO: 44)

(amino acid)

MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFT-(SEQ ID NO: 45)

Human NME7-A2 sequence optimized for E. coli expression:

(DNA)

atgaatcactccgaacgctttgtttttatcgccgaatggtatgacccgaatgcttccctgctgcgccgctacgaactgctgttttatccgggcgatggtagcgtggaaatgcatgacgttaaaaatcaccgtacctttctgaaacgcacgaaatatgataatctgcatctggaagacctgtttattggcaacaaagtcaatgtgttctctcgtcagctggtgctgatcgattatggcgaccagtacaccgcgcgtcaactgggtagtcgcaaagaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttctga (SEQ ID NO: 46)

(amino acid)

MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDA

Human NME7-A3 sequence optimized for E. coli expression:

(DNA)

atgaatcactccgaacgctttgtttttatcgccgaatggtatgacccgaatgcttccctgctgcgccgctacgaactgctgttttatccgggcgatggtagcgtggaaatgcatgacgttaaaaatcaccgtacctttctgaaacgcacgaaatatgataatctgcatctggaagacctgtttattggcaacaaagtcaatgtgttctctcgtcagctggtgctgatcgattatggcgaccagtacaccgcgcgtcaactgggtagtcgcaaagaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttacctga (SEQID NO: 48)

(amino acid)

MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICE

Human NME7-B sequence optimized for E. coli expression:

(DNA)

atgaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttctga (SEQID NO: 50)

(amino acid)

MNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF-(SEQ ID NO: 51)

Human NME7-B1 sequence optimized for E. coli expression:

(DNA)

atgaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttcaaaattctggataattga (SEQID NO: 52)

(amino acid)

MNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN-(SEQ ID NO: 53)

Human NME7-B2 sequence optimized for E. coli expression:

(DNA)

atgccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttctga (SEQID NO: 54)

(amino acid)

MPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF-(SEQ ID NO: 55)

Human NME7-B3 sequence optimized for E. coli expression:

(DNA)

atgccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttcaaaattctggataattga (SEQID NO: 56)

(amino acid)

MPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN-

Human NME7-AB sequence optimized for E. coli expression:

(DNA)

atggaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttcaaaattctggataattga (SEQID NO: 58)

(amino acid)

MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN- (SEQ ID NO: 59)

Human NME7-AB1 sequence optimized for E. coli expression:

(DNA)

Atggaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttctga (SEQID NO: 60)

(amino acid)

MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF- (SEQ ID NO: 61)

Mouse NME6

(DNA)

Atgacctccatcttgcgaagtccccaagctcttcagctcacactagccctgatcaagcctgatgcagttgcccacccactgatcctggaggctgttcatcagcagattctgagcaacaagttcctcattgtacgaacgagggaactgcagtggaagctggaggactgccggaggttttaccgagagcatgaagggcgttttttctatcagcggctggtggagttcatgacaagtgggccaatccgagcctatatccttgcccacaaagatgccatccaactttggaggacactgatgggacccaccagagtatttcgagcacgctatatagccccagattcaattcgtggaagtttgggcctcactgacacccgaaatactacccatggctcagactccgtggtttccgccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaggaaccccagctgcggtgtggtcctgtgcactacagtccagaggaaggtatccactgtgcagctgaaacaggaggccacaaacaacctaacaaaacctag (SEQID NO: 62)

(amino acid)

MTSILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRTRELQWKLEDCRRFYREHEGRFFYQRLVEFMTSGPIRAYILAHKDAIQLWRTLMGPTRVFRARYIAPDSIRGSLGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPVHYSP

Human NME6:

(DNA)

Atgacccagaatctggggagtgagatggcctcaatcttgcgaagccctcaggctctccagctcactctagccctgatcaagcctgacgcagtcgcccatccactgattctggaggctgttcatcagcagattctaagcaacaagttcctgattgtacgaatgagagaactactgtggagaaaggaagattgccagaggttttaccgagagcatgaagggcgttttttctatcagaggctggtggagttcatggccagcgggccaatccgagcctacatccttgcccacaaggatgccatccagctctggaggacgctcatgggacccaccagagtgttccgagcacgccatgtggccccagattctatccgtgggagtttcggcctcactgacacccgcaacaccacccatggttcggactctgtggtttcagccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaagagccccagttgcgctgtggccctgtgtgctatagcccagagggaggtgtccactatgtagctggaacaggaggcctaggaccagcctga (SEQID NO: 64)

(amino acid)

MTQNLGSEMASILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRC

Human NME6 1:

(DNA)

Atgacccagaatctggggagtgagatggcctcaatcttgcgaagccctcaggctctccagctcactctagccctgatcaagcctgacgcagtcgcccatccactgattctggaggctgttcatcagcagattctaagcaacaagttcctgattgtacgaatgagagaactactgtggagaaaggaagattgccagaggttttaccgagagcatgaagggcgttttttctatcagaggctggtggagttcatggccagcgggccaatccgagcctacatccttgcccacaaggatgccatccagctctggaggacgctcatgggacccaccagagtgttccgagcacgccatgtggccccagattctatccgtgggagtttcggcctcactgacacccgcaacaccacccatggttcggactctgtggtttcagccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaagagccccagttgcgctgtggccctgtgtga (SEQID NO: 66)

(amino acid)

MTQNLGSEMASILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRC

Human NME6 2:

(DNA)

Atgctcactctagccctgatcaagcctgacgcagtcgcccatccactgattctggaggctgttcatcagcagattctaagcaacaagttcctgattgtacgaatgagagaactactgtggagaaaggaagattgccagaggttttaccgagagcatgaagggcgttttttctatcagaggctggtggagttcatggccagcgggccaatccgagcctacatccttgcccacaaggatgccatccagctctggaggacgctcatgggacccaccagagtgttccgagcacgccatgtggccccagattctatccgtgggagtttcggcctcactgacacccgcaacaccacccatggttcggactctgtggtttcagccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaagagccccagttgcgctgtggccctgtgtga (SEQID NO: 68)

(amino acid)

MLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPV-(SEQ ID NO: 69)

Human NME6 3:

(DNA)

atgctcactctagccctgatcaagcctgacgcagtcgcccatccactgattctggaggctgttcatcagcagattctaagcaacaagttcctgattgtacgaatgagagaactactgtggagaaaggaagattgccagaggttttaccgagagcatgaagggcgttttttctatcagaggctggtggagttcatggccagcgggccaatccgagcctacatccttgcccacaaggatgccatccagctctggaggacgctcatgggacccaccagagtgttccgagcacgccatgtggccccagattctatccgtgggagtttcggcctcactgacacccgcaacaccacccatggttcggactctgtggtttcagccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaagagccccagttgcgctgtggccctgtgtgctatagcccagagggaggtgtccactatgtagctggaacaggaggcctaggaccagcctga (SEQID NO: 70)

(amino acid)

MLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPVCYSPEGGV

Human NME6 sequence optimized for E. coli expression:

(DNA)

atgacgcaaaatctgggctcggaaatggcaagtatcctgcgctccccgcaagcactgcaactgaccctggctctgatcaaaccggacgctgttgctcatccgctgattctggaagcggtccaccagcaaattctgagcaacaaatttctgatcgtgcgtatgcgcgaactgctgtggcgtaaagaagattgccagcgtttttatcgcgaacatgaaggccgtttcttttatcaacgcctggttgaattcatggcctctggtccgattcgcgcatatatcctggctcacaaagatgcgattcagctgtggcgtaccctgatgggtccgacgcgcgtctttcgtgcacgtcatgtggcaccggactcaatccgtggctcgttcggtctgaccgatacgcgcaataccacgcacggtagcgactctgttgttagtgcgtcccgtgaaatcgcggcctttttcccggacttctccgaacagcgttggtacgaagaagaagaaccgcaactgcgctgtggcccggtctgttattctccggaaggtggtgtccattatgtggcgggcacgggtggtctgggtccggcatga (SEQID NO: 72)

(amino acid)

MTQNLGSEMASILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRC

Human NME6 1 sequence optimized for E. coli expression:

(DNA)

atgacgcaaaatctgggctcggaaatggcaagtatcctgcgctccccgcaagcactgcaactgaccctggctctgatcaaaccggacgctgttgctcatccgctgattctggaagcggtccaccagcaaattctgagcaacaaatttctgatcgtgcgtatgcgcgaactgctgtggcgtaaagaagattgccagcgtttttatcgcgaacatgaaggccgtttcttttatcaacgcctggttgaattcatggcctctggtccgattcgcgcatatatcctggctcacaaagatgcgattcagctgtggcgtaccctgatgggtccgacgcgcgtctttcgtgcacgtcatgtggcaccggactcaatccgtggctcgttcggtctgaccgatacgcgcaataccacgcacggtagcgactctgttgttagtgcgtcccgtgaaatcgcggcctttttcccggacttctccgaacagcgttggtacgaagaagaagaaccgcaactgcgctgtggcccggtctga (SEQID NO: 74)

(amino acid)

MTQNLGSEMASILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRC

Human NME6 2 sequence optimized for E. coli expression:

(DNA)

atgctgaccctggctctgatcaaaccggacgctgttgctcatccgctgattctggaagcggtccaccagcaaattctgagcaacaaatttctgatcgtgcgtatgcgcgaactgctgtggcgtaaagaagattgccagcgtttttatcgcgaacatgaaggccgtttcttttatcaacgcctggttgaattcatggcctctggtccgattcgcgcatatatcctggctcacaaagatgcgattcagctgtggcgtaccctgatgggtccgacgcgcgtctttcgtgcacgtcatgtggcaccggactcaatccgtggctcgttcggtctgaccgatacgcgcaataccacgcacggtagcgactctgttgttagtgcgtcccgtgaaatcgcggcctttttcccggacttctccgaacagcgttggtacgaagaagaagaaccgcaactgcgctgtggcccggtctga (SEQID NO: 76)

(amino acid)

MLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPV-(SEQ ID NO: 77)

Human NME6 3 sequence optimized for E. coli expression:

(DNA)

atgctgaccctggctctgatcaaaccggacgctgttgctcatccgctgattctggaagcggtccaccagcaaattctgagcaacaaatttctgatcgtgcgtatgcgcgaactgctgtggcgtaaagaagattgccagcgtttttatcgcgaacatgaaggccgtttcttttatcaacgcctggttgaattcatggcctctggtccgattcgcgcatatatcctggctcacaaagatgcgattcagctgtggcgtaccctgatgggtccgacgcgcgtctttcgtgcacgtcatgtggcaccggactcaatccgtggctcgttcggtctgaccgatacgcgcaataccacgcacggtagcgactctgttgttagtgcgtcccgtgaaatcgcggcctttttcccggacttctccgaacagcgttggtacgaagaagaagaaccgcaactgcgctgtggcccggtctgttattctccggaaggtggtgtccattatgtggcgggcacgggtggtctgggtccggcatga (SEQID NO: 78)

(amino acid)

MLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPVCYSPEGG

Overall length OriGene-NME7-1

(DNA)

gacgttgtatacgactcctatagggcggccgggaattcgtcgactggatccggtaccgaggagatctgccgccgcgatcgccatgaatcatagtgaaagattcgttttcattgcagagtggtatgatccaaatgcttcacttcttcgacgttatgagcttttattttacccaggggatggatctgttgaaatgcatgatgtaaagaatcatcgcacctttttaaagcggaccaaatatgataacctgcacttggaagatttatttataggcaacaaagtgaatgtcttctctcgacaactggtattaattgactatggggatcaatatacagctcgccagctgggcagtaggaaagaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggccct tgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggataatacgcgtacgcggccgctcgagcagaaactcatctcagaagaggatctggcagcaaatgatatcctggattacaaggatgacgacgataaggtttaa (SEQ IDNO: 80)

(amino acid)

MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDNTRTRRLEQKLISEEDLAANDILDYKDDDDKV (SEQID NO: 81)

Abnova NME7-1 Total length

(amino acid)

MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN (SEQID NO: 82)

Abnova part NME7-B

(amino acid)

DRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKIL (SEQID NO: 83)

Histidine tag

(ctcgag) caccaccaccaccaccactga (SEQ ID NO: 84)

Strept II tag

(accggt) tggagccatcctcagttcgaaaagtaatga (SEQID NO: 85)

N-10 peptide:

QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 86)

C-10 peptide

GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDV (SEQ ID NO: 87)

Immune peptide derived from human NME7:

LALIKPDA (SEQ ID NO: 88)

MMMLSRKEALDFHVDHQS (SEQ ID NO: 89)

ALDFHVDHQS (SEQ ID NO: 90)

EILRDDAICEWKRL (SEQ ID NO: 91)

FNELIQFITTGP (SEQ ID NO: 92)

RDDAICEW (SEQ ID NO: 93)

SGVARTDASESIRALFGTDGIRNAA (SEQ ID NO: 94)

ELFFPSSGG (SEQ ID NO: 95)

KFTNCTCCIVKPHAVSEGLLGKILMA (SEQ ID NO: 96)

LMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVT (SEQ IDNO: 97)

EFYEVYKGVVTEYHD (SEQ ID NO: 98)

EIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNA (SEQID NO: 99)

YSGPCVAM (SEQ ID NO: 100)

FREFCGP (SEQ ID NO: 101)

VHCTDLPEDGLLEVQYFFKILDN (SEQ ID NO: 102)

IQNAVHCTD (SEQ ID NO: 103)

TDLPEDGLLEVQYFFKILDN (SEQ ID NO: 104)

PEDGLLEVQYFFK (SEQ ID NO: 105)

EIINKAGFTITK (SEQ ID NO: 106)

MLSRKEALDFHVDHQS (SEQ ID NO: 107)

NELIQFITT (SEQ ID NO: 108)
EILRDDAICEWKRL (SEQ ID NO: 109)

SGVARTDASESIRALFGTDGI (SEQ ID NO: 110)

SGVARTDASES (SEQ ID NO: 111)

ALFGTDGI (SEQ ID NO: 112)

NCTCCIVKPHAVSE (SEQ ID NO: 113)

LGKILMAIRDA (SEQ ID NO: 114)

EISAMQMFNMDRVNVE (SEQ ID NO: 115)

EVYKGVVT (SEQ ID NO: 116)

EYHDMVTE (SEQ ID NO: 117)

EFCGPADPEIARHLR (SEQ ID NO: 118)

AIFGKTKIQNAV (SEQ ID NO: 119)

LPEDGLLEVQYFFKILDN (SEQ ID NO: 120)

GPDSFASAAREMELFFP (SEQ ID NO: 121)

Immune peptide derived from human NME7

ICEWKRL (SEQ ID NO: 122)

LGKILMAIRDA (SEQ ID NO: 123)

HAVSEGLLGK (SEQ ID NO: 124)

VTEMYSGP (SEQ ID NO: 125)

NATKTFREF (SEQ ID NO: 126)

AIRDAGFEI (SEQ ID NO: 127)

AICEWKRLLGPAN (SEQ ID NO: 128)

DHQSRPFF (SEQ ID NO: 129)

AICEWKRLLGPAN (SEQ ID NO: 130)

VDHQSRPF (SEQ ID NO: 131)

PDSFAS (SEQ ID NO: 132)

KAGEIIEIINKAGFTITK (SEQ ID NO: 133)

Immune peptide derived from human NME1

MANCERTFIAIKPDGVQRGLVGEIIKRFE (SEQ ID NO: 134)

VDLKDRPF (SEQ ID NO: 135)

HGSDSVESAEKEIGLWF (SEQ ID NO: 136)

ERTFIAIKPDGVQRGLVGEIIKRFE (SEQ ID NO: 137)

VDLKDRPFFAGLVKYMHSGPVVAMVWEGLN (SEQ ID NO: 138)

NIIHGSDSVESAEKEIGLWFHPEELV (SEQ ID NO: 139)

KPDGVQRGLVGEII (SEQ ID NO: 140)

NME inhibition

Applicants have previously discovered that the key growth factor receptor MUC1 * and its activating ligand, the dimer form NM23-H1 (also referred to as NME1), mediates the growth of most solid tumor cancers. .. The inventor subsequently discovered that this same growth factor / growth factor receptor pair further mediates the growth of pluripotent stem cells. Enzymatic cleavage of MUC1 *, compatible splice variants or transmembrane proteins (MUC1) is found in all differentiated pluripotent human stem cells (Hikita, 2008 et al.) And in the majority of solid tumor cancers (Mahanta, 2008). Years) is expressed. When the stem cells differentiate, the cleavage of MUC1 subsides and MUC1 returns to its full-length inactive form. In stem cells and cancer cells, MUC1 * functions as a growth factor receptor. Ligand-induced dimerization of MUC1 * promotes cancer cell growth and survival, making cancer cells resistant to chemotherapeutic agents (Fessler, 2009 et al.). Inhibition of ligand-induced dimerization of the MUC1 * extracellular domain significantly inhibits cancer cell growth in vitro (Fig. 1) and in vivo (Fig. 2). In stem cells, ligand-induced dimerization of MUC1 * stimulates growth and survival while inhibiting differentiation. Both stem cells and cancer cells secrete NM23-H1. In the dimeric form, NM23-H1 dimerizes the extracellular domain of MUC1 * in order to proliferate stem cells and inhibit their differentiation.
The dimeric form of NME1 not only promotes stem cell growth and pluripotency, but also induces human stem cells to return to their earliest, most proliferative state, called the "naive" state ((Nichols J). , Smith A (2009); Hanna et al, 2010; Amit M, et al, 2000; Ludwig TE, et al 2006; Xu C, et al, 2005; Xu RH, et al, 2005; Smagghe et al 2013). To date, this is the only natural factor that has been shown to maintain human stem cells in a naive state, as they are present in the very inner assembly of their early embryos.

Here, the inventor has found that other molecules previously thought to be stem cell specific are also expressed in cancer cells. Growth factors and growth factor receptors that are expressed during embryogenesis but irregularly re-expressed in cancer cells are excellent therapeutic targets because disabling them has no significant negative effect on the patient. It becomes. Thus, in embryogenesis, we identified stem cell growth factors and receptors that are active in embryonic stem (ES) cells or induced pluripotent stem (iPS) cells but are irregularly reactivated in cancer cells. However, the development of therapeutic means that disable them or suppress their expression is a major advance in the art.

Applicants have more recently discovered that many genes and gene products expressed in human stem cells are also expressed in human cancer. For example, MUC1 *, an enzymatically cleaved form of an alternative spliced variant or transmembrane protein, MUC1 *, is expressed in all differentiated pluripotent human stem cells and in the majority of solid tumor cancers. When stem cells differentiate, MUC1 cleaves and MUC1 returns to its full-length inactive form. On stem and cancer cells, MUC1 * functions as a growth factor receptor. Dimerization of MUC1 * promotes stem and cancer cell growth; it inhibits stem cell differentiation and causes cancer cells to return to a less differentiated state. Both stem cells and cancer cells secrete NM23-H1 in dimeric form and dimerize the extracellular domain of MUC1 * to proliferate stem cells and inhibit their differentiation.

The present inventor has discovered that NME family members that are actively expressed in stem cells are irregularly upregulated in cancer cells. In addition to NM23-H1, other NME family proteins act as growth factors and transcriptional regulators that promote stem and cancer cell growth and inhibit their differentiation. When dimerized, NME1 promotes stem cell growth and pluripotency. The NME1 dimer also mediates the growth and dedifferentiation of MUC1-positive cancer cells. When stem cell concentration increases and more and more NME1 is secreted from the stem cells, NME1s form hexamers, which actually induce differentiation and stop pluripotent stem cell growth. It is known that cancer cells may appear less differentiated than normal adult cells. In fact, the degree to which cancer cells appear morphologically dedifferentiated is related to the degree of cancer activity. Therefore, treating cancer patients or patients at risk of developing cancer with hexamer-type NME1, which will cause cancer cell differentiation and limit their ability to self-replicate, is a valuable therapeutic approach. .. ..

In addition to NME1, NME6 and NME7 are expressed in early-stage stem and cancer cells. Western blot analysis was performed on a wide variety of human stem and cancer cell lines, indicating that both stem and cancer cells express and secrete NME1, NME6 and NME7. In one such experiment, for human embryonic stem cells (BGO1v) and human MUC1 * -positive breast cancer cells (T47D), the cell lysates were NME1 (NM23-H1), NME6 (data not shown) or NME7. The presence was inspected. Figures 3A and 3B show that NME1 and NME7 were easily detected in the lysates of stem cells and cancer cells. NME6 (~ 22 kDa) was detected by the more sensitive analysis shown in Figure 4D. Pull-down analysis was performed on stem cells and cancer cells using antibodies that bind to the cytoplasmic domain of MUC1. Western in Figures 3C and 3D shows that both NME1 and NME7 bind to MUC1 (because it is present in the stem and cancer cells). NME7 is produced in both stem cells and cancer cells, but the present inventor has found that it is present in cells as a full-length protein ~ 42 kDa. However, before being secreted, NME7 is cleaved. The secreted form appeared to lack its leader sequence DM10, confirming a clear molecular weight of ~ 33 kDa. FIG. 5 is a panel of photographs of Western blots of human embryonic stem (ES) cells (A) and induced differentiation pluripotent stem (iPS) cells (B, C) tested for the presence of NME7. Western blots show the presence of three types of NME7 in cell lysates. They have a clear molecular weight of ~ 42 kDa (overall), ~ 33 kDa (NME7-AB domain lacking N-terminal DH domain) and small ~ 25 kDa species. However, only lower molecular weight species are secreted in the conditioned medium (C).

The inventor has made several constructs for the expression of human NME7. One of these commonly expressed in E. coli was secreted in a soluble form as a monomer, and the NME1 dimer nearly functioned to promote stem cell growth, pluripotency and inhibition of differentiation. .. In this construct, the leader sequence "DM10" was removed from the sequence. It produced seeds of approximately the same molecular weight (33 kDa) as the secretory form of the protein. The protein was made as a histidine-tagged protein, first purified on an NTA-Ni column, and then purified by FPLC to 98% purity or higher. The present inventor refers to this type as NME7, NME7-AB. The present invention is not intended to be limited by the exact nature of the NME7 protein. The NME7-AB protein produced by the present inventor may simply be the smallest portion of the natural protein required for its stem / cancer promoting function.
The inventor has shown that NME7-AB functions in a manner that is essentially the same as NME7 processed in its natural state, as demonstrated in the Experimental and Examples contained herein. It was. However, the naturally occurring cleavage site of NME7 may differ from the starting site of the NME7-AB N-terminus by the present inventor. Inhibitors of NME7 can act on native proteins containing DM10 at the N-terminus, or can act to inhibit cleavage of NME7 to the secretory form. Figure 6A-C shows the FPLC trace (A) of NME7-AB, which is subsequently purified by a nickel column, the SDS-PAGE gel (B) of the unpurified protein and the FPLC trace (C) of the final product. Nanoparticle analysis was performed to show that NME7 as a monomer can simultaneously bind to two PSMGFR peptides (SEQ ID NO: 6) in the MUC1 * extracellular domain.
The histidine-tagged PSMGFR peptide was immobilized on NTA-SAM-coated nanoparticles. Recombinant NME7-AB, which was confirmed to be a monomer by FPLC and a natural gel and expressed lacking the N-terminal leader sequence of DM10, was added to the nanoparticles. The addition of NME7 changes the gold nanoparticle solution from pink to blue, which indicates that NME7 is simultaneously bound to two peptides on two separate nanoparticles. It brings the particles closer together, resulting in a characteristic color variation (Fig. 7). Another Elisa experiment demonstrated that the NME7 monomer dimerizes two MUC1 * extracellular domain peptides. The first PSMGFR peptide was coupled to BSA and immobilized on a multi-well plate. Recombinant NME7-AB was added. After an appropriate wash step, a biotin-modified second PSMGFR peptide was added. Subsequently, labeled streptavidin was added, which clearly showed that the NME7 monomer was able to bind two MUC1 * extracellular domain peptides at the same time (Fig. 8). These results indicate that NME7 via the two NDPK domains binds and dimerizes MUC1 * in stem and cancer cells.

NME7 functions much like the NME1 dimer. Like the NME1 dimer, NME7 fully supports the growth of human stem cells. Test panels of human stem cells (embryo "ES" and induced differentiation pluripotency "iPS") have the smallest serum-free basis with either NME7-AB or NME1 dimer added as a single growth factor or cytokine. It was cultured in medium. Stem cells grew faster than general FGF-containing medium growth, did not differentiate spontaneously, and were returned to a naive state, as evidenced by their presence of two active X chromosomes. 9 and 10 show photographs of day 1 and day 3 human HES-3 embryonic stem cells cultured in NME1 dimer or NME7-AB, respectively.
As can be clearly seen, stem cells appear to grow equally without any sign of differentiation. Note that naive stem cells do not grow in colonies, but rather in single layers that become sheets as density is achieved. FIG. 11 shows photographs of immunocytochemistry (ICC) experiments confirming that stem cells cultured in NME7-AB over 10 passages positively stain standard pluripotency markers. FIG. 12 shows a photograph of an ICC experiment confirming that stem cells cultured in NME7-AB are in a naive state. The cells of panel (A) were cultured in FGF on mouse feeder cells as is standard.
The stained antibody caused it to bind to concentrated trimethylated lysine 27 on histone 3 (H3K27me), resulting in a red dot; it is one X chromosome inactive (XaXi) and the stem cells "prime" (primary stimulation). It indicates that it has progressed to the "received)" state. The cells in panel (B) are the same cells as photographed in (A), except that they were subcultured in NME7-AB. As can be seen in the insert, the H3K27me antibody produced a cloud staining pattern, which showed that both X chromosomes were active (XaXa), demonstrating that the cells had returned to a naive state. Therefore, we have demonstrated that NME7 fully supports stem cell growth and pluripotency and also returns them to a later, less mature, naive or ground state than the primed state. ..

The inventor then sought to determine if NME7 is an active growth factor that further drives the growth of cancer cells. If so, therapeutic agents that block the interaction of NME7 with the MUC1 * extracellular domain can inhibit or prevent cancer growth in patients. Rabbit polyclone antibodies prepared for the NME7 A and B domains were added to T47D, MUC1 * positive breast cancer cells and cell growth was analyzed. Even at very low nanomolar concentrations, anti-NME7 inhibited the growth of cancer cells (Fig. 13-15). In a preferred embodiment, the therapeutic agent for the treatment of cancer is an antibody that binds to the NDPKA domain of NME7.
In a more preferred embodiment, the therapeutic agent is an antibody that binds to the NDPK B domain of NME7. In an even more preferred embodiment, the therapeutic agent is an antibody that binds to a sequence in the A or B domain of NME7 that is not present in NME1. In an even more preferred embodiment, the therapeutic agent is an antibody that inhibits the interaction between NME7 and MUC1 *. In the most preferred embodiment, the therapeutic agent is an antibody that inhibits the function of NME7 whose function promotes the growth of a cancer tumor or the return to a cancer-like state.

We refer to one way in which NME ligands function as growth factors by binding and dimerizing to the extracellular domain of MUC1 *. NME family proteins have one or more NDPK domains. These NDPK domains have catalytic functions that are independent of their function as growth factors and transcriptional regulators and are not required for them. NME family proteins bind to the extracellular domain of MUC1 * via their NDPK domain.

Different NME family proteins are expressed at different times during normal embryonic development. NME7 is the most primitive of the NME family of proteins that regulates stem cell growth and is expressed only in very early embryogenesis in vivo. NME7 is a single ~ 42 kDa protein with an N-terminal leader sequence called the DM10 domain and two NDPK domains, A and B. ELISA analysis shows that NME7 binds to and dimerizes the MUC1 * transmembrane receptor. Since NME7 has two NDPK domains, it is a pseudodimer capable of constantly dimerizing the MUC1 * receptor.

In contrast, NME1 has a molecular weight of approximately half that of NME7 (~ 17 kDa) and has only one NDPK domain. NME1 acts as a growth factor that promotes growth and inhibits differentiation only when it is a dimer. At higher concentrations, NME1 can form hex summers. In contrast to the dimer, NME1 hexamer induces differentiation. Thus, NME1 (which is later expressed during embryogenesis) has the ability to block itself, thereby limiting self-renewal. On the other hand, NME7 cannot. Wild-type (wt) NME1 exists at analytical concentrations, primarily as hex summers. Mutant NME1 proteins, such as S120G, which form stable dimeric aggregates, are isolated from cancer and therefore continuously activate the MUC1 * receptor. The inventor made recombinant NME1-wt and S120G mutants.
By altering the refolding protocol, we were able to stabilize aggregates that were essentially 100% hexsumers or 100% dimers. In addition, the inventor has isolated an aggregate of NME1-S120G, a mixture of dimers, tetramers and hexomers. FIG. 16 shows these various multimers on a natural gel. FIG. 17 (A) discloses a gel of the NME1 protein used in the SPR experiment (B), where the PSMGFR peptide of the MUC1 * extracellular domain was immobilized on the chip and different NME1 proteins crossed the surface. did. The results show that the dimeric form of NME1 is the type that binds to the MUC1 * extracellular domain. Panel (C) shows an experiment in which the PSMGFR peptide was attached to NTA-Ni-SAM coated nanoparticles and a recombinant NME1 dimer or hexamer was added to the nanoparticles.
If the interaction occurs, the gold nanoparticles turn blue, otherwise they remain pink. As can be seen, only the dimer binds to the MUC1 * peptide on the nanoparticles and dimerizes the two peptides of the two different nanoparticles, essentially cross-linking the particles. When added to solution, the MN-C2 anti-MUC1 * antibody Fab blocks the interaction between the NME1 dimer and the MUC1 * PSMGFR peptide. Panel (D) shows photographs of human stem cells cultured in a dimer, hexamer, or NME1 dimer (NM23) with a free PSMGFR peptide to competitively inhibit interaction.
As can be seen in the photo, only the NME1 dimer promotes pluripotent stem cell growth. In nature, differentiation is induced when the concentration of stem cells reaches a critical mass and their secretion of NME1 reaches the concentration at which they form hexamers. FIG. 18 is a cartoon depicting the mechanism supported by the experimental example of the present application. NME7 and NME1 thereby function for NME1 to control itself and promote pluripotency.

NME6 is also expressed during the very early development. NME6 is reportedly a sponge-like dimer of some sort. NME6 must be expressed at a sufficiently high level to form a dimer in order to be able to activate growth and inhibit differentiation. Therefore, it is expressed at a later stage than NME7. NME6 further binds to the PSMGFR peptide in the MUC1 * extracellular domain. Pull-down analysis showed that NME6 binds to MUC1 * in cancer and stem cells. We have created recombinant NME6 as a wild protein or with a single point-onset mutation S139G that mimics the S120G mutation that makes NME1 dimer-oriented. In addition, another NME6 mutant was created in this sensitive area so that human NME6 appears to be spongy NME6, which reportedly exists as a dimer.
These mutations are V142D and V143A, in addition to S139A. The ELISA analysis shown in Figures 19 A, B and C shows that NME6 binds to the PSMGFR peptide in the MUC1 * extracellular domain. In Part A, NME6-wt is purified as a monomer or high molecular weight multimer. The surface is coated with PSMGFRMUC1 * peptide, ELISA analysis shows preferential binding of NME6 monomer to MUC1 * peptide. In Part B, the NME6 multimer is dissociated by dilution in SDS. This ELISA shows that as the multimer is dissociated, binding to the MUC1 * peptide increases.
This figure shows that NME6-wt and two dimer-oriented mutants bind to the MUC1 * peptide. Figure 19 The gel in D-H shows the following expression: NME6-wt (D), NME6 (E), dimer with S139G mutation corresponding to mutant S120G, which increases dimer formation in human NME1. It has been reported that NME6 (F), which carries three mutations that mimic the human form of the spongy form, and a single-stranded protein (GH) that links two NME6 proteins. Panel I is a pull-down analysis using antibodies against the cytoplasmic tail of MUC1 showing that NME6 binds to MUC1 in cancer and stem cells.
Therefore, effective anti-cancer agents are antibodies, small molecules or other agents that block the binding of NME6 dimers to MUC1 * extracellular domain peptides. In a preferred embodiment, the therapeutic agent for cancer is an antibody that binds to the NDPKA domain of NME6. In a more preferred embodiment, the therapeutic antibody binds to a sequence of NME6 that is not present in NME1.

Human stem cells that mimic the embryonic stem cells of the inner cell mass of the blastocyst, where they are just the earliest stem cells, are called "naive" stem cells. Until recently, researchers have been unable to maintain or generate genetically unmodified naive human stem cells in vitro. The present inventor genetically engineered unmodified human stem cells in the naive state by culturing the cells in NME1 dimer or NME7 and in the absence of other growth factors or cytokines, especially in the absence of bFGF. Recently succeeded in generating a gene. Furthermore, the inventor has shown that these naive stem cells proceed to a more mature "primed" state as soon as they are exposed to bFGF.
To demonstrate that NME7 is expressed at very high levels in very early stage stem cells, we have either cultured in either NME1 or NME7 (naive) or bFGF (primed). Western blot analysis was performed on human stem cells cultured in, and then the presence of NME7 was examined. Primed embryonic stem cells, which are more differentiated than naive stem cells, are expressed only at trace amounts of NME7. In a pristine comparison, early "naive" (also called "ground") stem cells express high levels of NME7 (Fig. 3B, compare lane 1 "naive" to lane 2 "prime"). .. NME7 is expressed in cancer cells at levels comparable to its expression in early-stage stem cells (Fig. 3B, compare lane 3 “cancer cells” to lane 1 “naive stem cells”). For this reason, NME7 and NME6 can be neutralized without significant therapeutic side effects, as their primary role is in early development rather than in adulthood.

NME7 is a single molecule with two NDPK domains and therefore functions as an NME1 dimer in one embodiment. One of NME7's cohesive partners is MUC1 *. Our studies show that NME7 binds and dimerizes the extracellular domain of MUC1 * -positive cells in order to promote growth and inhibit the differentiation of both human stem cells and cancer cells. NME7 is also detected in cancer cells, conditioned medium, cytoplasm and nuclei, indicating that it functions as a secreted growth factor and also as a transcriptional regulator that binds DNA directly or indirectly. Western blots of FIG. 20 show that both NME1 and NME7 have both cytoplasms and nuclei of human cancer cells (T47D), embryonic stem cells (BGO1v and HES-3) and induced pluripotent stem (iPS) cells. Indicates that it is present in.
These data indicate that NME1 and NME7 can function directly or indirectly to influence gene transcription. Thus, in one aspect of the invention, the function of NME1 or NME7 is inhibited by the addition of a potentially small molecule drug that inhibits the binding of NME1 or NME7 to DNA, and transcription of NME1 or NME7. Drugs that inhibit function are anticancer drugs that can be administered to cancer patients or patients at risk of developing cancer.

The NME protein is probably expressed at different levels in different cancer cells. Most cancers are MUC1 * positive and show high expression of NME1, NME6 and NME7. DU145 prostate cancer cells express more NME7 than NME1 or NME6. PC3 prostate cancer cells (they are MUC1 * negative) did not have detectable NME1 or NME7, but had high expression of NME6 (Figure 21).

NME7 exists in different types.

NME7 is expressed as a different species. Some of these species are specific for cancer cells. The full-length NME7 is 42 kDa and is composed of two different NDPK domains and one DM10 leader sequence at its N-terminus. The full-length NME7 can be found in the cytoplasm. A species of NME7 of ~ 33 kDa, which is consistent with a species composed of NDPK A and B domains, but lacks the DM10 leader sequence, is found exclusively in conditioned media for both stem and cancer cells. (Fig. 5 and Fig. 22). Note that these findings are independent of the dimeric recombinant NME1 added to culture stem cells. FIG. 23 shows that nothing was detected when the gel of FIG. 22 was exposed and retested for the presence of the histidine tag on the recombinant protein.
These results indicate that the smaller molecular weight NME7 is the secreted growth factor type. We have created an NME7 mutant with a molecular weight of ~ 33 kDa, called NME7-AB, which contains the NDPK A and B domains but does not have the DM10 domain. This recombinant NME7-AB can fully support the growth of pluripotent human stem cells in serum-free medium lacking other growth factors or cytokines. NME7-AB more fully supported the growth of MUC1 * -positive cancer cells. These experiments showed that the secretory form of NME7 was a growth factor form, which contained the NDPK A and B domains, lacked most or all DM10, and had a molecular weight of ~ 33 kDa.
FIG. 24 shows various cell lysates and corresponding adjusted cells tested for the presence of NME7 using mouse monoclonal antibody (A) or simply another monoclonal antibody (B) that simply recognizes the N-terminal DM10 sequence. A photograph of a Western blot of the medium is shown. Lack of binding of DM10-specific antibodies to ~ 33 kDa NME7 species in samples from the prepared medium of the cells is largely due to the secreted form of NME7, but not all of the N-terminal DM10 leader sequences. Indicates that is missing.

Another smaller ~ 25kDa NME7 species is also present from time to time. Western blots show the presence of lower molecular weight species ~ 25 kDa from the beginning. This ~ 25 kDa NME7 contains the NDPK A domain and has a single binding site for MUC1 *. The ~ 25 kDa band was excised and analyzed by mass spectrometry. Mass specs have shown that ~ 25 kDa species are essentially composed of the NDPKA domain.

It has been reported that NME7 is expressed in other human tissues even at low levels. However, the inventor has discovered that it is the secreted form of NME7 that functions as a growth factor. Also, some adult tissues can express NME7, but an important aspect is whether it is secreted. A stem cell that expresses and secretes NME7 is a stem cell that is earlier-more differentiated than a stem cell that expresses or does not secrete NME7, and earlier-thus more differentiated than a stem cell that does not secrete NME7. ..
Cancer cells that express and secrete NME7 are less differentiated and more aggressive than cancer cells that do not secrete NME7. Therefore, analysis of NME7 and secreted NME7 levels can be used to predict tumor activity, planned treatment, therapeutic monitoring effects, and to stratify patient populations for clinical trials.
Therefore, antibodies that detect NME1, NME6 or NME7 can be used to detect the appearance of cancer or as a diagnostic tool to assess cancer activity, where high levels of NME1, NME6 or NME7 Is associated with tumor activity and inadequate consequences. High levels of NME7 and NME6 are particularly associated with tumor activity and therefore poor prognosis. Patient samples that can be tested with antibodies against NME1, NME6 or NME7 can be samples of body fluids including blood, tissue biopsy, needle biopsy and the like.

NME family proteins can function to promote cancer.

The inventor has previously reported that the NME protein promotes embryonic and iPS cell growth and pluripotency in the same way that it leads to the return of cells to a stem-like state. Since many of the genetic features of the stem-like state and the cancer state are shared, the inventor further concludes that NME family proteins can induce the cancer state. In a preferred embodiment, the NME family protein is 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85 relative to NME1 or NME1. %, 90%, 95% or 97% or more NME proteins with sequence identity. Here, the protein is a dimer. In a more preferred embodiment, the NME family protein is 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70 relative to at least one of NME7 or NME7 domain A or B. It is an NME protein with a%, 75%, 80%, 85%, 90%, 95% or 97% or higher sequence identity and can dimerize the MUC1 * growth factor receptor.

Here, the inventor reports that the dimeric NME1, the dimeric bacterium NME1, NME7 or NME7-AB was effective for:
a) Fully support the growth and pluripotency of human ES or iPS while inhibiting differentiation;
b) Return somatic cells to a more stem-like or more cancer-like state;
c) Convert cancer cells, also called tumor-inducing cells, into a highly metastatic cancer stem cell state.

The inventor has high sequence homology to human NME1 and is a recombinant bacterial NME found in Porphyromonas gingivalis W83 and Halomonas Sp593 (“HSP 593”), which have been reported to be present in a dimeric state. Proteins were prepared (FIGS. 25 and 26). HSP 593 is well expressed in E. coli, and an important part is present as a dimer, after which the aggregate was purified by FPLC to confirm the dimer aggregate (Fig. 25A). A direct binding experiment was performed showing that "Bacterial NME from Halomonas Sp. 593 binds to the PSMGFR peptide in the MUC1 * extracellular domain" (Fig. 25B). The sequence alignment between HSP 593 and human NME1 or human NME7 domain A or B shows that the bacterial NME bound to the MUC1 * extracellular domain is 40-41% identical to human NME1 and human NME7-A, and NME7. -B shows that it was 34% identical (Fig. 27A-C).

Additional experiments have been conducted showing that bacterial NMEs with a human NME1 or NME7 identity of more than 30%, preferably 40% or more, exert a human NME-like function that promotes cancer and stem cell growth and survival. Was done. Many of the bacterial NMEs that had this high sequence identity to human NMEs were reported to be associated with human cancers. Therefore, we have tested the idea of whether many bacteria induce cancer or worsen existing cancers in humans. Bacterial NME was functionally analyzed for human NME1 and NME7. Human HES-3 embryonic stem cells were cultured in serum-free minimal basal medium with HSP 593, human NME1 dimer or human NME7-AB as the only growth factor or cytokine. Since human NME1 and NME7 fully supported human stem cell growth, they also supported HSP 593-derived bacterial NME (Figure 28 A-F compared with Figures 9 and 10).

Human NME1 dimer or human NME7 can return somatic cells to a less mature state expressing markers for stem and cancer cells. HSP 593-derived bacterial NMEs have been tested with human homologues to determine if it can mimic their function by being able to direct somatic cells to a cancer-like state. Human fibroblasts were cultured in serum-free minimal basal medium with HSP 593, human NME1 dimer or human NME7-AB as the only growth factor or cytokine. RT-PCR analysis showed that, like human NME, the bacterium NME1 HSP 593 had turned somatic cells into an OCT4 positive state by the 19th.
Recalling that stem cells and metastatic cancer cells may grow anchorage-independently, we repeated the experiment. However, this time, a raw kinase inhibitor was added to the set of cells to attach the cells to the surface. When floating cells were forced to attach to the surface, RT-PCR showed that there was actually a 7-fold increase in the stem / cancer marker OCT4, and a 12-fold increase in the stem / cancer marker Nanog. It was as high as the increase in (Fig. 30).
Experimental photographs showed dramatic morphological variation, as fibroblasts reverted when cultured in human or bacterial NME (Fig. 31-38). The relative order of efficiency of returning somatic cells to a less mature state was NME7> NME1 dimer> bacterial NME1. Reportedly, the transcriptional regulator BRD4 and cofactor JMJD6 suppress NME7 and upregulate NME1 (Lui With et al, 2013). We have found that these factors are expressed at lower levels in naive stem cells than they were in late-stage primed stem cells (Fig. 39).
This result activates stem cell growth as a dimer, but when the cell secretes more and it forms hexamar s, the hexamar s does not bind MUC1 * and differentiation is induced. We support the hypothesis that NME7 is a stem cell growth factor expressed earlier than NME1 because it cannot interrupt or control self-renewal in the manner that NME1 does.

Chromatin rearrangement factors MBD3 and CHD4 have recently been reported to block the induction of pluripotency (Rais Yet al, 2013). RT-PCR analysis of human fibroblasts grown in human NME1 or NME7 or bacterial NME1 shows that the NME protein suppresses all four pluripotent (BRD4, JMJD6, MBD3 and CHD4) blockers (BRD4, JMJD6, MBD3 and CHD4). Figure 40). Synthetic graphs of RT-PCR experiments show that the relative ability to increase pluripotent genes and decrease pluripotent blockers is NME7> NME1> HSP 593 NME. However, apparently, bacterial NME from HSP593 upregulates expression of human NME7 and NME1 (FIGS. 41 and 42). Therefore, the NME1 dimer, NME7 and bacterial NME1 dimer allow somatic cells to return to a less mature cancer / stem-like state.

Another function of NME1 and NME7 is the ability to mutate cancer cells into a more metastatic cancer stem cell state, also called tumor-inducing cells. Cancer cell test panels were cultured in serum-free minimal basal medium with human NME7-AB NME1 dimer or human NME1 dimer (“NM23” in the figure) as the only growth factor or cytokine. .. After a few days in this medium, the cells began to float from the surface and continued to grow in solution. "Floating matter" was collected and analyzed separately by PCR. Cells in the other wells were treated with a raw kinase inhibitor ("Ri in the figure).
Quantitative PCR analysis shows an increase in cancer stem cell markers. Some of them were considered and used only as stem cell markers (Miki J et al 2007, Jeter CR et al 2011, Hong X et al 2012Faber A et al 2013, Mukherjee Det al 2013, Herreros-Villanueva M et al, 2013, Sefah K et al, 2013; Su HT et al 2013). Figures 44-47 show that culturing with NME protein causes cancer cells to return to highly metastatic tumor-inducing cells, upregulated to more than 200-fold with metastatic receptor CXCR4 and up to 200-fold with SOX2. It is rated and shows up to 10-fold increase in E-cadherin (CDH1), NANOG and MUC1.
In conclusion, cancer cells secrete NME7 and NME1 that activate MUC1 and upregulate the host of cancer and cancer stem cell genes. We observed an increase in cancer stem cell markers and metastasis even in MUC1-negative cells, more moderately but pronely (Fig. 47). Thus, we see that NME7 can enter these cells, perhaps by routes other than MUC1 *, where it can still act as a transcriptional regulator and affect the expression levels of these genes. I concluded. As a hex summer, NME1 must be a dimer to function in this way, as it does not activate stem or cancer growth. However, many cancers mutate NME1 which resists the formation of self-renewal-restricted hex summers. Unlike NME1, NME7 is always active.

2i inhibitors, which are small molecule inhibitors of the MAP kinase signaling pathway MEK and GSK3 beta, have been reported to return mouse primed stem cells to a naive state (Silva J et al, 2008). .. The inventor also suspected whether these inhibitors could also bring human cancer cells back to the cancer stem cell state. T47D breast cancer cells are in serum-free minimal basal medium with the added 2i inhibitor, in the same basal medium with the added human recombinant NME7-AB, or in the same basal medium with both NME7-AB and 2i. , Was cultured for 10 days.
The results shown in FIG. 48 show that the cancer stem cell marker E-cadherin (CDH1), metastatic receptor CXCR4, as well as the stem / cancer markers OCT4, SOX2 and NANOG, are 2i, 2i + NME7-AB. , NME7-AB alone is shown to be highly upregulated. The relative performance of inducing cancer stem cell markers was NME7> NME7 + 2i> 2i (Fig. 48).
When cancer cells were treated with either 2i or NME7-AB, the expression of pluripotency-inhibiting chromatin regulators and transcriptional regulators BRD4, JMJD6, MBD3 and CHD4 was similarly down-regulated (Fig. 49). ..

Therefore, a drug that disables any of the following functions of NME1 dimer, human or bacteria, or NME7 is a potent anticancer drug and can be administered to patients for the treatment or prevention of cancer; Ability to promote stem cell growth, bind to the MUC1 * peptide PSMGFR, return somatic cells to a less mature state, and mutate cancer cells to a cancer stem cell state.

In support of the idea that NME inhibitors are potent anti-cancer agents, we conducted an experiment to apply it to many other antibodies that bind to NME7, NME7-AB, as well as other NME proteins. I was convinced that it could be expanded. The present inventor grew MUC1 * -positive cancer cells in the presence or absence of the rabbit polyclone antibody produced against human NME7. Tumor cell growth decreased significantly in a concentration-dependent manner, as shown in Figures 13-15. Polyclone anti-NME7 is not an ideal anti-cancer agent in that it is an antibody produced by rabbits. For therapeutic agents, monoclonal antibodies are selected based on their ability to generate monoclonal antibodies and specifically inhibit cancer cell growth, and ideally block the binding of NME7 to the MUC1 * extracellular domain. .. Finally, human or humanized antibodies can be selected.

NME Function 1: One way the NME protein exerts its cancer-promoting function is by binding to a clip-type MUC1 transmembrane protein, called MUC1 * in the present invention, consisting primarily of PSMGFR sequences. Dimerization of the MUC1 * extracellular domain stimulates the growth and dedifferentiation of stems and cancer cells.

NME Function 2: Another way in which NME proteins exert their function of promoting cancer, dedifferentiation, pluripotency, growth or survival, directly or indirectly, to stimulate or suppress other genes. Transferring them to a functioning nucleus. It was previously reported that OCT4 and SOX2 bind to the promoter sites of MUC1 and its cleavage enzyme MMP16 (Boyer et al, 2005). The same study reported that SOX2 and NANOG bind to the promoter site of NME7. Based on our experiments, we conclude that these "Yamanaka" pluripotency factors (Takahashi and Yamanaka, 2006) upregulate MUC1, its cleavage enzyme MMP16 and its activation ligand NME7. Attached.
BRD4 suppresses NME7, while its cofactor JMJD6 upregulates NME1 (determined by the present inventor), a self-regulating stem cell growth factor that is expressed later than NME7 during embryogenesis. It was previously reported elsewhere (Thompson et al). More recently, siRNA suppression of Mbd3 or Chd4 significantly reduced resistance to iPS production (Rais Y et al 2013 et al.). Experimental evidence from the present inventor has shown that there is a mutual feedback loop in which NME7 suppresses BRD4 and JMJD6 while suppressing inhibitors of pluripotent Mbd3 and CHD4. The inventor noted that in naive human stem cells, these four factors BRD4, JMJD6, Mbd3 and CHD4 are suppressed compared to their expression in later stages of primed stem cells. .. Furthermore, the present inventor also noted that a 2i inhibitor (inhibitor of Gsk3β and MEK) that returns mouse primed stem cells to a naive state downregulates the same four factors BRD4, JMJD6, Mbd3 and CHD4. ..

Furthermore, the present inventor has found that NME7 upregulates SOX2 (> 150X), NANOG (~ 10X), OCT4 (~ 50X), KLF4 (4X) and MUC1 (10X). Importantly, we have shown that NME7 upregulates cancer stem cell markers, including CXCR4 (~ 200X) and E-cadherin (CDH1). NME7 is the most primitive stem cell growth and pluripotency mediator, and it is a potent factor in mutating somatic cells into cancerous states as well as mutating cancer cells into more metastatic cancer stem cells. Much evidence has led to the conclusion that there is. FIG. 50 is a map of the interaction of NME7 with relevant regulators of stem / cancer status as evidenced by the experiments of this application.
The dimer NME1 functioned much like NME7 in its ability to transform somatic cells into stem / cancer-like states and, to a lesser extent, mutate cancer cells into metastatic cancer stem cells. Similarly, bacterial NME dimers with high homology to human NME1 or NME7, such as Halomonas Sp 593, are completely, like NME1 dimers and NME7 monomers, human stem cell growth, pluripotency and It was able to support survival, cancer cell growth and survival, returning somatic cells to a cancer / stem cell state and mutating cancer cells to more metastatic cancer stem cells.

The present inventors can therefore support human stem cell growth, pluripotency and survival, cancer cell growth and survival, return somatic cells to a cancer / stem cell state, and mutate cancer cells into more metastatic cancer stem cells. We have come to the conclusion that drugs that can abolish the function of NME proteins are ideal targets for anticancer treatment. Therapeutic agents here either disable the NME protein, block its binding to MUC1 *, block its function as a direct or indirect transcription factor, or block the function as described above. Is.
In a preferred embodiment, the agent that blocks the function of the NME protein is an antibody. In another preferred embodiment, the agent blocks the function or dimerization of the NME1 dimer. In an even more preferred embodiment, the drug blocks the function of NME7. The antineoplastic agent that blocks the function of one of these NME proteins can be an alternative nucleic acid. For example, nucleic acids that inhibit the expression of NME, such as sh- or siRNA, antisense nucleic acids, are exemplified. Alternatively, the drug may indirectly suppress the expression of NME. For example, increased expression of BRD4 suppresses NME7 and thus acts as an anti-cancer agent.
In another embodiment, the agent that inhibits the function of the targeted NME protein can be a small molecule synthetic chemical that acts directly on or inhibits the expression of the NME protein. Separately or in combination, these agents are powerful anti-cancer agents for the treatment or prevention of cancer.

In one case, the drug that inhibits the targeted NME protein is an antibody, an anti-cancer drug that is administered directly to the patient for the treatment or prevention of cancer. In a preferred embodiment, the primary cancer or its progeny is a MUC1 * positive cancer. The antibody can be the original antibody or a modified antibody-like molecule. Antibodies or antibody-like molecules can be linked to cytotoxic substances that activate the immune response or substances that activate the immune response.
For example, the anti-NME antibody portion can be modified to be part of a therapeutic molecule as described in CAR (Chimeric Antigen Receptor) T Cell Technology (Porter D et al, 2011). The antibody can be divalent, monovalent, bispecific humanized or partially humanized. Antibody or antibody-like molecule, including those used by TillerT et al (2013), binding analysis in vitro, using phage display techniques and others such Ylanthia ^(TM) were randomized such as the system It can be produced using a humanized antibody epitope library.

In another aspect of the invention, the agent that inhibits the targeted NME protein is an antibody produced by the patient. Here, the patient is immunized with a portion of the targeted NME protein so that the patient initiates an immune response containing anti-NME antibodies. Such immunization is performed, for example, as a vaccine, for the treatment or prevention of cancer.

In another aspect, the invention comprises the identification of peptide sequences derived from MUC1 *, NME1 humans, bacteria NME1 and NME7 that give rise to antibodies that are anti-cancer agents. These peptide sequences can be used to generate therapeutic antibodies, as well as vaccines, nucleic acid sequences for antisense type therapy, methods for identifying cancer-inducing bacteria, diagnostic methods and drug screening methods. it can. In one aspect of the invention, the peptides of the sequences described herein can be supplemented with an adjuvant or in cancer by inducing antibody production against the targeted NME protein in a patient in a host animal or human. As a vaccine to immunize against, it can be fused to other peptides used to stimulate the immune system and produce anti-cancer antibodies.
In a preferred embodiment, the targeted NME protein is a bacterial NME with 30% or more sequence identity to human NME1 or NME7 domain A or B. In a more preferred embodiment, the targeted NME protein is human NME1, where the antibody comprises NME1 having a dimer formation oriented mutation, such as an S120G mutation, a P69S mutation or a C-terminal cleavage. Can be specifically targeted. In an even more preferred embodiment, the targeted NME protein is NME7 (SEQ ID NO: 13), which substantially comprises a cleaved form as described as NME7-AB (SEQ ID NO: 39).

Transgenic mice expressing human NME7, human NME1 or dimerization-oriented mutants or bacterial NMEs are used in drug discovery to grow cancer cells in vivo and as an element of anti-cancer vaccines of NME-derived peptides. It will be of great help to test the immunization effect. For example, Murid NME proteins differ from human NME proteins. Mouse stem cells grow using a single growth factor, LIF. On the other hand, LIF cannot support the growth of human stem cells. The inventor now knows that cancer cells and stem cells grow by a similar mechanism. Therefore, transplanting human cancer cells into mice poses a problem in addition to the immune response in mice to human cancer cells; that is, mice are growth factors that alone promote cancer growth and mutation into cancer stem cells. Does not produce certain dimer NME1 or human NME7.

We have found that animals injected with human NME7 develop cancer more easily than mice not injected. For example, some cancer cells are very difficult to engraft in animals. The present inventor increased the engraftment rate of cancer cells several times by injecting human NME7 or NME7-AB into animals. Immune-damaged mice were injected with T47D breast cancer cells mixed with either Matrigel or NME7-AB at 50 / 50V / V. After 10 days, mice injected with NME7 mixed cells were further infused daily with NME7-AB (Fig. 51).
The group (dotted line) further infused with NME7-AB had larger tumors that grew at an accelerated rate. If NME7-AB is mixed with cancer cells at the time of infusion, and if mice are infused every 24 or 48 hours after infusion, engraftment rate, reduced cell count required and faster tumors Growth has been brought about. The range of cancer cell ratios to NME7 or the NME7 injection schedule is expected to vary from one mouse species to another and from one tumor type to another. With improvements in this method, genetically modified animals for human NME7 or NME7-AB greatly increase the engraftment rate of cancer cells, resulting in a decrease in the number of cells required to grow into a tumor in the animal. .. This allows the growth of primary patient cancer cells in animals expressing human NME7 or NME7-AB.

In one case, the cancer cells are injected into the animal and the animal is dosed with NME7 or NME7-AB. In a preferred embodiment, the animal is a genetically modified animal that expresses human NME7-AB. In a preferred embodiment, the cancer cells are primary cells from the patient. In this way, the animal, which can be a mouse, provides NME growth factor that allows the patient's cancer cells to return to a less mature, more metastatic state. In one embodiment, the host animal is infused with a candidate drug or compound and efficacy is assessed to predict the patient's response to treatment with the candidate drug or compound.
In another example, a first-line treatment or drug that has been administered to a patient or is being considered for the treatment of a patient is administered to an animal carrying the patient's cancer cells that have been brought back to a less mature state. Will be done. First-line therapy probably affects mutations that cancer cells adapt to avoid first-line therapy. After treatment, cancer cells can then be removed from the host animal and analyzed and characterized to identify mutations that occur in response to a treatment. Alternatively, the cancer cells can be left in the host animal, which is treated with another therapeutic agent to determine which agent inhibits or kills resistant cells or cancer stem cells.

Our experiments have shown that the differences between murine NME proteins and human NME proteins are largely due to the extremely inefficient engraftment of human cancer cells in mice. Injecting recombinant human NME7 into mice at the time of cancer cell injection significantly increased the rate of tumor engraftment and the rate of tumor growth. Therefore, transgenic mice expressing human NME7 or more preferably human NME7-AB can engraft patient cells in a mouse model for drug discovery, medication testing, or how the patient's cancer cells undergo drug therapy. It will allow it to be determined whether it will progress or mutate accordingly, and will significantly increase the rate of tumor engraftment.
For example, to avoid potential problems with NME7 expression during animal development, it would be advantageous to have human NME7 on an inducible promoter. Alternatively, cancer cells, including patient cells, are bacteria NME, NME7, or NME12 that mimic human NME such that the cells are mutated into cancer stem cells that require only 50-200 cells to induce a tumor in the animal. Can be cultured in a body. These cells can then be tested in vivo or in vitro, including transgenic animals carrying NME7, NME1 dimer, bacterial NME or a single chain of NME1 pseudodimers.

Recombinant animals expressing human NME (specifically NME7-AB) evaluate which immune peptides can be safely used to generate antibodies against NME1, bacterial NME and NME proteins, including NME7. Will help. For example, transgenic mice for human NME1, NME7 or NME7-AB could be immunized with one or more of the immune peptides described as in Figure 62-64, Peptide No. 1-53. Control mice were analyzed to confirm that anti-NME antibody was produced. Human tumor cells are then injected into transgenic mice, where inducible promoters induce expression of human NME protein in host animals. The efficacy and potential toxicity of the immune peptide were then increased in tumor engraftment, tumor growth rate and tumor of cells transplanted into transgenic mice compared to mice or control mice in which the inducible NME promoter was not activated. It is evaluated by comparing the evoked ability.
Toxicity determines bone marrow cell regeneration response time, number and type of circulating blood cells and overall bone marrow number in response to treatment with cytotoxic agents to bone marrow cells, as well as heart, liver and others. Evaluated by examination of organs such as. Immunopeptides derived from those listed in Peptide No. 1-53, Figure 62-64, which significantly reduce tumor-inducing capacity, tumor engraftment, or tumor growth rate with tolerable side effects, are anti-cancer treatments, prevention , Or as a vaccine, outside the patient or in humans, as an immune peptide for the production of antibodies.

Therefore, mice or other mammals that will spontaneously form tumors or will respond more like humans to the drug being tested, or will better allow human tumor engraftment. Animals are produced by the use of any one of many methods of introducing human genes into animals. Such methods are often cited as knock-in, knockout, CRISPR, TALEN and others. The present invention can be envisioned to use any method of expressing human NME7 or NME7-AB in mammals.
NME7 or NME7-AB can be induced by one of many methods of controlling the expression of known transgenes. Alternatively, the expression or timing of expression of NME7 can be controlled by the expression of another gene that is naturally expressed by the mammal. For example, it may be suitable for NME7 variants or NME7 expressed in tissues such as the heart. The NME7 gene is then operably linked to the expression of heart-expressed proteins such as MHC. In this example, expression of NME7 is possible when and where the MHC gene product is expressed. Similarly, it may be desirable to express human NME6 or NME7 in the prostate, for example, where the location and timing of expression is controlled by the expression of a prostate-specific protein. Similarly, in non-human mammals, human NME6 or NME7 expression can be controlled by genes expressed in breast tissue. For example, in transgenic mice, human NME6 or human NME7 is expressed from the prolactin promoter, or a similar gene.

Inhibitor of NME protein as an anticancer agent

Which NME protein is targeted by an inhibitor acting as an anticancer agent can depend on the type of cancer. For example, tumors that have been shown to carry a highly sequence-homologous bacterial NME to a human NME1 or NME7-A or -B domain or a human NME1 dimer or a bacterial NME that mimics the function of NME7-AB are bacterial NMEs. It will be treated with drugs or antibodies that inhibit the ability to target and dimerize proteins, bind to MUC1 *, or promote cancer growth or mutate cancer cells to cancer stem cells. Alternatively, in some cancer cells, the dimerization-oriented NME protein can be irregularly reactivated or mutated to resist the formation of its hexamer form. In addition, other cancers can irregularly reactivate expression of NME7 or cleaved NME7-AB. Therefore, therapeutic antibodies that recognize NME1, NME1 dimers and / or the bacterial NME that mimics NME7-AB may be useful in the prevention or treatment of cancer. Alternatively, diagnostic analysis is performed to determine which NME inhibitors are effective against the cancer or cancer subset.

Antibodies that bind to NME7 and inhibit its tumorigenicity are potent anti-cancer agents and can be administered to patients for the treatment or prevention of cancer. Antibodies that inhibit the tumorigenicity of NME7 or NME7-AB are antibodies that inhibit the ability of NME7 to bind to its cognate binding partner, which in one case is the PSMGFR portion of the MUC1 * receptor. Is. In other cases, NME7 can function by entering cells, migrating to the nucleus and acting as a direct and indirect transcriptional regulator, XCR4, SOX2, MUC1, E-cadherin, OCT4, NME7 or NME7- Activation in genes that promote tumorigenesis, such as AB, downregulates BRD4, JMJD6, MBD3 and CHD4, all of which result in increased tumorigenic potential of the cell. Therefore, cancer therapeutic or prophylactic antibodies, when tested in vitro or in vivo, inhibit NME7 binding to MUC1 * or NME7 or CXCR4, SOX2, MUC1, E-cadherin, OCT4, BRD4, It is like inhibiting the binding of its cofactor to the nucleic acid promoter site of JMJD6, MBD3 or CHD4. These antibodies can be administered to patients with cancer or at risk of developing cancer.
As is well known in the art, antibodies and antibody-like molecules can be produced using all NME1, NME6 or NME7 proteins. Alternatively, peptides or portions of the protein can be used. In addition, in other methods, the peptide will be injected into the host animal with a carrier molecule or an adjuvant to elicit an immune response. Antibodies can then be obtained from animals in standard ways, including monoclonal antibodies produced by antibody-producing cells, obtained from animals that can be humanized.
The present invention can also use NME1, NME6 or NME7 proteins, or peptides from which their sequences are derived, in screening analysis. In one such example, antibody libraries can be screened for their ability to bind NME1, NME6 or NME7, where antibodies that bind to the targeted NME protein treat or prevent cancer. Used for. Moreover, the library need not be composed of the antibody itself. A library of antibody epitopes or fragments is screened for binding to a portion of the NME protein to identify therapeutic antibodies for the treatment of cancer patients or those at risk of developing cancer.
One or more of the immunogenic peptides listed in Figures 62-64 identify and select antibody epitopes that can be later modified into antibody-like molecules for antibody production in host animals or for administration to patients. Ideal for. In another embodiment, the peptide whose sequence is derived from NME1, NME6 or preferably NME7 is administered directly to humans. It produces an immune response in which the administered human produces an immune response, including antibody production, as a cancer vaccine. One or more of the peptides listed in Figure 62-64 (SEQ ID NO. 88-140) are suitable for antibody production or selection, which is suitable for use as a vaccine for antibody production in host animals. It is also used as a diet for screening libraries of synthetic peptides or antibody epitopes, such as the Ylanthia ^{Registered Trademark System.} Here, the antibody will inhibit cancer by inhibiting the function of NME7 or by marking it for degeneration. In another embodiment, the invention is directed to peptide fragments of NME family proteins, which are selected from SEQ ID NOS: 88-140, preferably 88-133, more preferably 88-121, into NME7. Used to generate or select anti-cancer antibodies or antibody epitopes that bind and inhibit.

NME7 may be an ideal therapeutic target for the treatment or prevention of cancer, as its major role appears to be in very early embryogenesis and is not expressed at significant levels in adult tissues. Therefore, drugs that disable NME7 are expected to block and inhibit cancer in healthy adult tissues with minor side effects. Our studies show that cancer cells and naive stem cells secrete NME7, which can function as their only required growth factor.
In addition, metastatic cancer cells, cell aggregates (which are cancer stem cells or tumor-inducing cells), produce them as NME1 dimers, bacterial NME dimers, or NME7 (where NME7 produces many cancer stem cells). ), The present inventor has shown that the increase is preferential. Therefore, agents that disable the NME protein are excellent anti-cancer therapeutic agents, and are particularly useful for inhibiting or preventing cancer stem cells or tumor-inducing cells. In a preferred embodiment, the NME protein targeted by the therapeutic agent is human or bacterial NME1, where the therapeutic agent inhibits dimerization, inhibits binding to MUC1 *, or such as CXCR4. Inhibits the ability to upregulate various pluripotent genes or cancer stem cell genes. In a more preferred embodiment, the NME protein targeted by the therapeutic agent is NME7, where the therapeutic agent inhibits NME7 expression, inhibits NME7 binding to MUC1 *, or cleaves the DM10 domain. Or inhibits its ability to upregulate pluripotent genes such as CXCR4 or cancer stem cell genes.

Therefore, targeted therapeutic agents that inhibit the growth and dedifferentiation of cancer cells are agents that abolish NME7 function. The NME7 functions that therapeutic agents disable for the treatment of cancer are not limited to, but include:
1) Ability to bind to MUC1 * extracellular domain;
2) Ability to bind to DNA;
3) Ability to promote stem cell proliferation;
4) Ability to inhibit differentiation;
5) Ability to act as a transcriptional regulator;
6) Ability to be secreted by cells.

Drugs that abolish NME7 function, such as those listed above, are not limited to: but include: antibodies, chemicals, small molecules, microRNAs, antisense nucleic acids, inhibitory RNAs, RNAi, siRNAs. In one example, the therapeutic agent is an antibody, which is an antibody-like substance that contains monovalent, bivalent, bispecific, multiclonal, monoclonal, or regions that mimic the variable domains of those antibodies. sell. In another example, the therapeutic agent is a chemical, such as a small molecule. Drugs that suppress NME7, such as RNAi or siRNA, can also be applied as anti-cancer treatments. In a preferred embodiment, these agents block the interaction of NME7 with the extracellular domain of MUC1 *.

In the alternative approach, BRD4 suppresses NME7, so drugs that upregulate BRD4 are given to patients for the treatment or prevention of cancer.

Immune NME peptides to produce therapeutic antibodies

To date, little is known about NME proteins and their functions, especially newly identified NME proteins such as NME7. Until recently, NME1 was considered a hex summer. The crystal structures of NME1 and NME2 as hex summers have been published (Webb PA et al, 1995; Min Ket al, 2002), but there is information on how the NME dimer or NME7 folds. Almost not provided. However, the present inventor is based on the published hexamer structure of NME1, sequence alignment in human NME1, human NME7 and bacterial NME, which can mimic human NME1 and NME7, especially Halomonas Sp.593. Specific peptide sequences were identified from Halomonas Sp.593, human NME7 and human NME1, which are predicted to give rise to therapeutic antibodies for the treatment or prevention of known cancers.

FIG. 61 is a sequence alignment between human NME1 and human NME7-A or -B domains. FIG. 27 is a sequence alignment between bacterial NMEs from human NME1 and Halomonas Sp 593 and between bacterial NMEs from human NME7-A or -B domains and Halomonas Sp. 593 (“HSP 593”).

Peptides 1-34 listed in FIG. 62 with SEQ ID NOS: 88-121 are peptides from human NME7 selected for their low homology to human NME1.
NME7 peptides 35-46 (SEQ ID NOS: 122-133) (Fig. 63) are reasonably unique sequences for regions of NME7 that appear structurally important due to protein integrity or ability to bind to MUC1 * peptides. It was selected because it is there. Both sets of NME7 sequences are expected to give rise to antibodies that bind to NME7. However, a second set of NME7 peptides may function to disable the ability to bind to NME7 or MUC1 * peptides. These peptides are expected to recognize NME7 or human NME1 or bacterial NME, resulting in antibodies that can be used to treat or prevent cancer.

Peptides 47-53 (SEQ ID NOS: 134-140) listed in FIG. 64 are human NME1 sequences selected for their high sequence homology to both human NME7 and bacterial HSP 593 NME. It is inferred that it is important for the structure or the binding to MUC1 *. These peptides are expected to give rise to antibodies capable of recognizing NME1, NME7 or bacterial NME and can be used in the treatment or prevention of cancer.

Listed as peptides 1-34 (SEQ ID NOS: 88-121) in FIG. 62, they have low homology to human NME1, but high homology to human NME7-A or NME7-B. The peptide it has must give rise to an antibody that is oriented towards binding to human NME7, which plays a role in adult tissues, except in cases such as cancer tissues where it is desired to inhibit its activity. Should be restricted.

Peptides 35-46 (SEQ ID NOS: 122-133) listed in FIG. 63 are peptide sequences from NME7, which are sequence homology, published crystal structure of NME1 hexomer, and C-terminal excision. Is oriented towards dimerization and is assumed to be important for structural integrity or MUC1 * binding, based on the knowledge that it does not inhibit MUC1 * binding or protein function in stem and cancer growth.
Peptides 47-53 (SEQ ID NOS: 134-140) listed in FIG. 64 are peptide sequences from NME1, which are sequence homology, published crystal structure of NME1 hexmer, and C-terminal excision. Is oriented towards dimerization and is assumed to be important for structural integrity or MUC1 * binding, based on the knowledge that it does not inhibit MUC1 * binding or protein function in stem and cancer growth.
Antibodies generated from peptides or peptide mimetics containing these sequences will produce antibodies that can be administered to patients for the treatment or prevention of cancer. Peptides or peptide mimetics containing these sequences constitute a cancer therapeutic vaccine that can generate antibodies at the host and thus be administered to a patient for the treatment or prevention of cancer.

Diagnostic analysis

Yet another aspect of the invention is diagnostic analysis, in which the patient's cancer or the patient's predominant NME at risk of developing cancer is split into NME1, bacterial NME, or full-length NME7 or NME7-AB types. Decide if it is a good one. Diagnostic analysis includes standard analysis such as IHC, ICC, FISH, RNA-Seq and other detection or sequencing techniques. However, unlike standard cancer diagnostic tests, analysis is performed to determine if NME1, NME7, or bacterial NME is present in greater amounts than those analyzed in the control group.
Antibodies that specifically inhibit or disable the presence of NME proteins in patients or in patient groups based on such determination of the type of NME protein expressed by the patient's cancer or by the cancer subset that afflicts many patients. NEM antibodies or other NME incapacitating agents are selected and administered to the patient. Similarly, diagnostic analysis shows whether a patient's NME protein has a protein dimerization-oriented mutation, and if so, a drug that disables that particular mutant NME for the treatment or prevention of cancer. It is used to determine if it is administered to a patient.

Antibodies that render the targeting NME protein incapacitated, or its similar receptor MUC1 *, preferentially target cancer cells, but not or to a lesser extent stem or ancestral cells. Further screening is performed to identify antibodies that do not. MUC1 is cleaved into MUC1 * type by various cleaving enzymes, and which enzyme cleaves MUC1 depends on the tissue type or the timing of cell or organism development.
For example, MMP14 is expressed at higher levels in stem cells than it is in breast cancer cells (Fig. 52). Conversely, MMP14 and ADAM17, as well as MUC1 cleaving enzymes, are expressed in DU145 prostate cancer cells 3 and 5 times higher than they are in human stem cells; in T47D breast cancer cells, MMP16 and ADAM17 make them stem cells. Twice as high as it is (Figs. 53 and 54).
In fact, when DU145 prostate cancer cell-injected mice were treated with the anti-MUC1 * antibody MN-E6 Fab, tumor growth was significantly inhibited (Fig. 55) and MMP14 and ADAM17 expression was reduced (Fig. 55). 56), MUC1 cleavage was reduced, and expression of microRNA-145, which signals differentiation, was increased (Fig. 57 A, B). Thus, MUC1 * can be altered at its distal N-terminus by more than 10 amino acids.
The C-terminus of MUC1 is intracellular and its N-terminus is extracellular. Our experiments show that the NME1 dimer binds to the N-10 version of the PSMGFR peptide. That is, removing the first 10 amino acids of the PSMGFR peptide, which corresponds to the majority of MUC1 * extracellular domains, does not affect the ability of the NME1 dimer to bind to the MUC1 * peptide. Antibodies that preferentially bind to the N-10 peptide (SEQ ID NO: 86) preferentially bind to MUC1 * because it is present in cancer cells. Conversely, antibodies that preferentially bind to the C-10 peptide (SEQ ID NO: 87) preferentially bind to stem and bone marrow cells rather than cancer cells (Fig. 58-60).
Thus, antibodies that target MUC1 * for the treatment or prevention of cancer can be produced by immunization with the PSMGFR peptide, N-10 peptide or C-10 peptide. Alternatively, therapeutic or antibody-like molecules for the treatment or prevention of cancer appear in cancer cells as opposed to those in stems and ancestral cells, so by selecting those that bind to MUC1 *. Can be identified.
In a preferred embodiment, the antibody is oriented towards binding to the N-10 peptide. In an even more preferred embodiment, the therapeutic antibody is selected by its ability to bind cancer cells rather than stem or ancestral cells. In one case, the antibody was first selected by ELISA or similar direct binding analysis for its ability to bind PSMGFR peptide, N-10 peptide or C-10 peptide, followed by various types of MUC1 * positive cancer cells. It was confirmed that it can be combined with. Here one antibody can bind to prostate cancer cells better than breast cancer cells, or vice versa, supporting the hypothesis of different cleavage sites of different tissue types.
The hybridoma supernatant is then coated on a multi-well plate. And the stem cells were sown on it. Since the stem cells are non-adherent, the wells covered with the antibody bound to the stem cells (or ancestral cells) attached the stem cells. On the other hand, the antibody to which no stem cells were attached was selected as the preferred anti-MUC1 * antibody for the treatment or prevention of cancer.

Thus, in another aspect, the invention directs a method of classifying cancer or stratifying cancer patients or suspected cancer patients, including the following steps:
(i) Analyzing patient samples for the presence of stem or ancestral cell genes or gene products;
(ii) Grouping of patients who share similar expression or expression levels of stem or ancestral cell genes or gene products.
In this way, patients can then be treated with agents that inhibit their stem or ancestral cell genes or gene products.

In other cases, the expression level of the stem or ancestral cell gene or gene product is analyzed to assess the severity of the cancer, where the expression or higher expression of the gene or gene product that is characteristic of the early stem or ancestral state. Expression indicates a more aggressive cancer, and expression or higher expression of a gene or gene product that is characteristic of later ancestral states indicates less aggressive cancer. Such decisions will then allow physicians to design strategies in treating more aggressive or non-aggressive cancer patients.

These methods of classifying or stratifying cancers can be performed with blood samples, body fluids or biopsies. Genes or gene products whose high expression levels indicate highly aggressive cancers include NME1, more preferably NME6, and even more preferably NME7.

The scope of rights of the present invention is not limited by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the above description and accompanying drawings. Such modifications are intended to be included within the scope of the claims of the present application. The following examples are presented as illustrations of the invention rather than limiting disclosure.

Example 1-Composition of minimal serum-free basal medium ("MM") (500 mls)

400ml DME / F12 / GlutaMAXI (Invitrogen # 10565-018)
100ml Knockout Serum Replacement (KO-SR (Invitrogen # 10828-028))
5ml100x MEM Non-essential Amino Acid Solution (Invitrogen # 11140-050)
0.9 ml (0.1 mM) β-mercaptoethanol (55 mM stock, Invitrogen # 21985-023)

Example 2-Testing for the presence of NME1, NME6 and NME7 in cancer and stem cells

In this series of experiments, we examined the expression of NME6 and NME7 in stem and cancer cells. In addition, we have identified MUC1 * as a target for NME7. To determine the presence or absence of NME1, NME6 and NME7, we first performed a Western blot analysis on the cell lysates. In Figure 3A, lysates from BGO1v human embryonic stem cells cultured in NME1 dimer on an anti-MUC1 * antibody-coated surface (Lane 1) or in bFGF on a MEFs surface (Lane 2). , T47D human breast cancer cell lysate (Lane 3), or NME1-wt as a positive control, was isolated by SDS-PAGE and then tested with anti-NME1-specific antibodies. The results show that NME1 is strongly expressed in human ES cells, even if cultured in NME1 dimer or bFGF, and T47D cancer cells. The same cell lysates were separated by SDS-PAGE and then tested with anti-NME6-specific antibody (anti-NME6 from Abnova). NME6 was not detected (data not shown), but it was subsequently detected in a more concentrated sample (see Figure 4).

In Figure 3B, the same cell lysates were separated by SDS-PAGE and then tested with anti-NME7 specific antibody (nm23-H7 B9 from Santa Cruz Biotechnology). The results show that NME7 is strongly expressed in human ES cells cultured in NME1 dimer on the surface of anti-MUC1 * antibody (Lane 1) and weakly expressed in the same ES cells cultured in bFGF on MEF. It shows that (Lane 2) and that it is strongly expressed in breast cancer cells (Lane 3).
Lane 4, to which NME1 was added, is blank, indicating that the NME7 antibody does not cross-react with NME1.
We have shown that they are expression markers that indicate that they are more naive than cells cultured in bFGF, that NME7 is significantly more expressed in stem cells cultured in NME1 dimer. The fact is that NME7 is expressed at higher levels in naive cells than in primed cells.

The inventor performed a pull-down analysis to determine whether NME7 also functions as its target receptor, MUC1 *, and as a growth factor. In these experiments, synthetic MUC1 * extracellular domain peptides (His-tagged PSMGFR sequences) were immobilized on the NTA-Ni magnetic bed. These beds were lysed from BGO1v human embryonic stem cells cultured in NME1 dimers on anti-MUC1 * antibody-coated surfaces (Lane 1) or in bFGF on MEFs surfaces (Lane 2). Incubated with T47D human breast cancer cell lysate (Lane 3). The bed was rinsed and the captured protein was released by the addition of imidazole.
Proteins were separated by SDS-PAGE and then tested with anti-NME1 antibody (Fig. 3C), anti-NME6 antibody (data not shown) or NME7 antibody (Fig. 3D). The results show that NME7 binds to the MUC1 * extracellular domain peptide. This indicates that in stem cells and cancer cells, NME7 activates the pluripotent pathway by dimerizing the MUC1 * extracellular domain via that portion of the two NDPK domains.

Example 3-Generation of proteinogenic constituents

Generation of recombinant NME7:
The first construct was prepared to make recombinant NME7 that can be efficiently expressed in soluble form. The first approach was to create a construct encoding a native NME7 (-1) or an alternative splicing variant NME7 (-2) with an N-terminal deletion. In some cases, the construct carried a strep tag or histidine tag to aid in its purification. NME7-1 was poorly expressed in E. coli, and NME7-2 was not expressed in E. coli. However, the target sequence was deleted and a new construct was created in which NME7 essentially contained the NDPK A and B domains with a calculated molecular weight of 31 kDa.
This new NME7-AB was very well expressed in E. coli and was present as a soluble protein. NME-A, a component in which the single NDPK domain was expressed, was not expressed in E. coli. NME7-AB was first purified on an NTA-Ni column and then further purified on a Sephadex 200 column by molecular sieving chromatography (FPLC). The purified NME7-AB protein was then tested for its ability to promote pluripotency and inhibit stem cell differentiation.

Example 4-Functional study of human recombinant NME7-AB

Testing of recombinant NME7 for its ability to maintain pluripotency and inhibit differentiation
A soluble variant of NME7, NME7-AB, was produced and purified. Human stem cells (iPS cat # SC101a-1 and system bioscience) were grown according to manufacturer's instructions in 4 nanograms / ml bFGF on a layer of mouse fibroblast feeder cells for 4 passages. ^{These source stem cells were then planted in 6-well cell culture plates (Vita TM} , Thermo Fisher) coated with 12.5 μg / well of monoclonal anti-MUC1 * antibody (MN-C3). Cells were planted at a density of 300,000 cells per well.
The basal medium was the smallest stem cell medium consisting of:
400 ml DME / F12 / GlutaMAX I (Invitrogen # 10565-018), 100 ml knockout serum substitute (KO-SR, Invitrogen # 10828-028), 5 ml 100xMEM non-essential amino acid solution (Invitrogen # 11140-050) and 0.9 ml (Invitrogen # 11140-050) 0.1 mM) β-mercaptoethanol (55 mM stock Invitrogen # 21985-023). The basal medium can be any medium.
In a preferred embodiment, the basal medium is free of other growth factors and cytokines. Either 8nM of NME7-AB or 8nM NM23-H1 was added to the basal medium, and the mixture was folded and purified as a stable dimer. Medium was changed every 48 hours and had to be recovered by accelerated growth and was subcultured on day 3 of planting.
Figures 9 and 10 show a daily comparison of growth with the NM23-H1 dimer to growth with the NME7 monomer. Both the NME7 and NM23-H1 (NME1) dimers grew pluripotently and were not differentiated, even in 100% confluence. As can be seen in the photograph, NME7 cells grew faster than cells growing in the NM23-H1 dimer. Regarding the number of cells in the initial recovery, it was confirmed that the NME7 culture produced 1.4 times more cells than the NM23-H1 dimer culture. ICC staining for a typical pluripotent marker confirmed that NME7-AB fully supported human stem cell growth, pluripotency and resistant differentiation (Fig. 11).

Example 5-Generation of mutants of NME6 and NME7

The following novel NME6 and NME7 mutants were designed and generated:
Human NM23-H7-2 sequence optimized for E. coli expression:

(DNA)

atgcatgacgttaaaaatcaccgtacctttctgaaacgcacgaaatatgataatctgcatctggaagacctgtttattggcaacaaagtcaatgtgttctctcgtcagctggtgctgatcgattatggcgaccagtacaccgcgcgtcaactgggtagtcgcaaagaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatact ttttcaaaattctggataattga (SEQID NO: 20)

(amino acid)

MHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN- (SEQID NO: 21)

Human NME7-A:

(DNA)

atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgtttttttga (SEQID NO: 22)

(amino acid)

MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFF-(SEQ ID NO: 23)

Human NME7-A1:

(DNA)

atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttacttga (SEQID NO: 24)

(amino acid)

MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFT-(SEQ ID NO: 25)

Human NME7-A2:

(DNA)

atgaatcatagtgaaagattcgttttcattgcagagtggtatgatccaaatgcttcacttcttcgacgttatgagcttttattttacccaggggatggatctgttgaaatgcatgatgtaaagaatcatcgcacctttttaaagcggaccaaatatgataacctgcacttggaagatttatttataggcaacaaagtgaatgtcttttctcgacaactggtattaattgactatggggatcaatatacagctcgccagctgggcagtaggaaagaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgtttttttga (SEQID NO: 26)

(amino acid)

MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDA

Human NME7-A3:

(DNA)

atgaatcatagtgaaagattcgttttcattgcagagtggtatgatccaaatgcttcacttcttcgacgttatgagcttttattttacccaggggatggatctgttgaaatgcatgatgtaaagaatcatcgcacctttttaaagcggaccaaatatgataacctgcacttggaagatttatttataggcaacaaagtgaatgtcttttctcgacaactggtattaattgactatggggatcaatatacagctcgccagctgggcagtaggaaagaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttacttga (SEQID NO: 28)

(amino acid)

MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRD

Human NME7-B:

(DNA)

atgaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttctga (SEQID NO: 30)

(amino acid)

MNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF-(SEQ ID NO: 31)

Human NME7-B1:

(DNA)

atgaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggataattagtga (SEQID NO: 32)

(amino acid)

MNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN-(SEQ ID NO: 33)

Human NME7-B2:

(DNA)

atgccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttctga (SEQID NO: 34)

(amino acid)

MPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF-(SEQ ID NO: 35)

Human NME7-B3:

(DNA)

atgccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggataattagtga (SEQID NO: 36)

(amino acid)

MPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN-(SEQ ID NO: 37)

Human NME7-AB:

(DNA)

atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggataattagtga (SEQID NO: 38)

(amino acid)

MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN - (SEQ ID NO: 39)

Human NME7-AB1:

(DNA)

atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttctga (SEQID NO: 40)

(amino acid)

MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF- (SEQ ID NO: 41)

Human NME7-A sequence optimized for E. coli expression:

(DNA)

atggaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttctga (SEQID NO: 42)

(amino acid)

MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFF-(SEQ ID NO: 43)

Human NME7-A1 sequence optimized for E. coli expression:

(DNA)

atggaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttacctga (SEQID NO: 44)

(amino acid)

MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFT-(SEQ ID NO: 45)

Human NME7-A2 sequence optimized for E. coli expression:

(DNA)

atgaatcactccgaacgctttgtttttatcgccgaatggtatgacccgaatgcttccctgctgcgccgctacgaactgctgttttatccgggcgatggtagcgtggaaatgcatgacgttaaaaatcaccgtacctttctgaaacgcacgaaatatgataatctgcatctggaagacctgtttattggcaacaaagtcaatgtgttctctcgtcagctggtgctgatcgattatggcgaccagtacaccgcgcgtcaactgggtagtcgcaaagaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttctga (SEQID NO: 46)

(amino acid)

MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDA

Human NME7-A3 sequence optimized for E. coli expression:

(DNA)

atgaatcactccgaacgctttgtttttatcgccgaatggtatgacccgaatgcttccctgctgcgccgctacgaactgctgttttatccgggcgatggtagcgtggaaatgcatgacgttaaaaatcaccgtacctttctgaaacgcacgaaatatgataatctgcatctggaagacctgtttattggcaacaaagtcaatgtgttctctcgtcagctggtgctgatcgattatggcgaccagtacaccgcgcgtcaactgggtagtcgcaaagaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttacctga (SEQID NO: 48)

(amino acid)

MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICE

Human NME7-B sequence optimized for E. coli expression:

(DNA)

atgaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttctga (SEQID NO: 50)

(amino acid)

MNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF-(SEQ ID NO: 51)

Human NME7-B1 sequence optimized for E. coli expression:

(DNA)

atgaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttcaaaattctggataattga (SEQID NO: 52)

(amino acid)

MNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN-(SEQ ID NO: 53)

Human NME7-B2 sequence optimized for E. coli expression:

(DNA)

atgccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttctga (SEQID NO: 54)

(amino acid)

MPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF-(SEQ ID NO: 55)

Human NME7-B3 sequence optimized for E. coli expression:

(DNA)

atgccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttcaaaattctggataattga (SEQID NO: 56)

(amino acid)

MPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN-

Human NME7-AB sequence optimized for E. coli expression:

(DNA)

atggaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttcaaaattctggataattga (SEQID NO: 58)

(amino acid)

MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN- (SEQ ID NO: 59)

Human NME7-AB1 sequence optimized for E. coli expression:

(DNA)

Atggaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttctga (SEQID NO: 60)

(amino acid)

MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF- (SEQ ID NO: 61)

Mouse NME6

(DNA)

Atgacctccatcttgcgaagtccccaagctcttcagctcacactagccctgatcaagcctgatgcagttgcccacccactgatcctggaggctgttcatcagcagattctgagcaacaagttcctcattgtacgaacgagggaactgcagtggaagctggaggactgccggaggttttaccgagagcatgaagggcgttttttctatcagcggctggtggagttcatgacaagtgggccaatccgagcctatatccttgcccacaaagatgccatccaactttggaggacactgatgggacccaccagagtatttcgagcacgctatatagccccagattcaattcgtggaagtttgggcctcactgacacccgaaatactacccatggctcagactccgtggtttccgccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaggaaccccagctgcggtgtggtcctgtgcactacagtccagaggaaggtatccactgtgcagctgaaacaggaggccacaaacaacctaacaaaacctag (SEQID NO: 62)

(amino acid)

MTSILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRTRELQWKLEDCRRFYREHEGRFFYQRLVEFMTSGPIRAYILAHKDAIQLWRTLMGPTRVFRARYIAPDSIRGSLGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPVHYSP

Human NME6:

(DNA)

Atgacccagaatctggggagtgagatggcctcaatcttgcgaagccctcaggctctccagctcactctagccctgatcaagcctgacgcagtcgcccatccactgattctggaggctgttcatcagcagattctaagcaacaagttcctgattgtacgaatgagagaactactgtggagaaaggaagattgccagaggttttaccgagagcatgaagggcgttttttctatcagaggctggtggagttcatggccagcgggccaatccgagcctacatccttgcccacaaggatgccatccagctctggaggacgctcatgggacccaccagagtgttccgagcacgccatgtggccccagattctatccgtgggagtttcggcctcactgacacccgcaacaccacccatggttcggactctgtggtttcagccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaagagccccagttgcgctgtggccctgtgtgctatagcccagagggaggtgtccactatgtagctggaacaggaggcctaggaccagcctga (SEQID NO: 64)

(amino acid)

MTQNLGSEMASILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRC

Human NME6 1:

(DNA)

Atgacccagaatctggggagtgagatggcctcaatcttgcgaagccctcaggctctccagctcactctagccctgatcaagcctgacgcagtcgcccatccactgattctggaggctgttcatcagcagattctaagcaacaagttcctgattgtacgaatgagagaactactgtggagaaaggaagattgccagaggttttaccgagagcatgaagggcgttttttctatcagaggctggtggagttcatggccagcgggccaatccgagcctacatccttgcccacaaggatgccatccagctctggaggacgctcatgggacccaccagagtgttccgagcacgccatgtggccccagattctatccgtgggagtttcggcctcactgacacccgcaacaccacccatggttcggactctgtggtttcagccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaagagccccagttgcgctgtggccctgtgtga (SEQID NO: 66)

(amino acid)

MTQNLGSEMASILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRC

Human NME6 2:

(DNA)

Atgctcactctagccctgatcaagcctgacgcagtcgcccatccactgattctggaggctgttcatcagcagattctaagcaacaagttcctgattgtacgaatgagagaactactgtggagaaaggaagattgccagaggttttaccgagagcatgaagggcgttttttctatcagaggctggtggagttcatggccagcgggccaatccgagcctacatccttgcccacaaggatgccatccagctctggaggacgctcatgggacccaccagagtgttccgagcacgccatgtggccccagattctatccgtgggagtttcggcctcactgacacccgcaacaccacccatggttcggactctgtggtttcagccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaagagccccagttgcgctgtggccctgtgtga (SEQID NO: 68)

(amino acid)

MLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPV-(SEQ ID NO: 69)

Human NME6 3:

(DNA)

Atgctcactctagccctgatcaagcctgacgcagtcgcccatccactgattctggaggctgttcatcagcagattctaagcaacaagttcctgattgtacgaatgagagaactactgtggagaaaggaagattgccagaggttttaccgagagcatgaagggcgttttttctatcagaggctggtggagttcatggccagcgggccaatccgagcctacatccttgcccacaaggatgccatccagctctggaggacgctcatgggacccaccagagtgttccgagcacgccatgtggccccagattctatccgtgggagtttcggcctcactgacacccgcaacaccacccatggttcggactctgtggtttcagccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaagagccccagttgcgctgtggccctgtgtgctatagcccagagggaggtgtccactatgtagctggaacaggaggcctaggaccagcctga (SEQID NO: 70)

(amino acid)

MLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPVCYSPEGGV

Human NME6 sequence optimized for E. coli expression:

(DNA)

Atgacgcaaaatctgggctcggaaatggcaagtatcctgcgctccccgcaagcactgcaactgaccctggctctgatcaaaccggacgctgttgctcatccgctgattctggaagcggtccaccagcaaattctgagcaacaaatttctgatcgtgcgtatgcgcgaactgctgtggcgtaaagaagattgccagcgtttttatcgcgaacatgaaggccgtttcttttatcaacgcctggttgaattcatggcctctggtccgattcgcgcatatatcctggctcacaaagatgcgattcagctgtggcgtaccctgatgggtccgacgcgcgtctttcgtgcacgtcatgtggcaccggactcaatccgtggctcgttcggtctgaccgatacgcgcaataccacgcacggtagcgactctgttgttagtgcgtcccgtgaaatcgcggcctttttcccggacttctccgaacagcgttggtacgaagaagaagaaccgcaactgcgctgtggcccggtctgttattctccggaaggtggtgtccattatgtggcgggcacgggtggtctgggtccggcatga (SEQID NO: 72)

(amino acid)

MTQNLGSEMASILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRC

Human NME6 1 sequence optimized for E. coli expression:

(DNA)

Atgacgcaaaatctgggctcggaaatggcaagtatcctgcgctccccgcaagcactgcaactgaccctggctctgatcaaaccggacgctgttgctcatccgctgattctggaagcggtccaccagcaaattctgagcaacaaatttctgatcgtgcgtatgcgcgaactgctgtggcgtaaagaagattgccagcgtttttatcgcgaacatgaaggccgtttcttttatcaacgcctggttgaattcatggcctctggtccgattcgcgcatatatcctggctcacaaagatgcgattcagctgtggcgtaccctgatgggtccgacgcgcgtctttcgtgcacgtcatgtggcaccggactcaatccgtggctcgttcggtctgaccgatacgcgcaataccacgcacggtagcgactctgttgttagtgcgtcccgtgaaatcgcggcctttttcccggacttctccgaacagcgttggtacgaagaagaagaaccgcaactgcgctgtggcccggtctga (SEQID NO: 74)

(amino acid)

MTQNLGSEMASILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRC

Human NME6 2 sequence optimized for E. coli expression:

(DNA)

Atgctgaccctggctctgatcaaaccggacgctgttgctcatccgctgattctggaagcggtccaccagcaaattctgagcaacaaatttctgatcgtgcgtatgcgcgaactgctgtggcgtaaagaagattgccagcgtttttatcgcgaacatgaaggccgtttcttttatcaacgcctggttgaattcatggcctctggtccgattcgcgcatatatcctggctcacaaagatgcgattcagctgtggcgtaccctgatgggtccgacgcgcgtctttcgtgcacgtcatgtggcaccggactcaatccgtggctcgttcggtctgaccgatacgcgcaataccacgcacggtagcgactctgttgttagtgcgtcccgtgaaatcgcggcctttttcccggacttctccgaacagcgttggtacgaagaagaagaaccgcaactgcgctgtggcccggtctga (SEQID NO: 76)

(amino acid)

MLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPV-(SEQ ID NO: 77)

Human NME6 3 sequence optimized for E. coli expression:

(DNA)

Atgctgaccctggctctgatcaaaccggacgctgttgctcatccgctgattctggaagcggtccaccagcaaattctgagcaacaaatttctgatcgtgcgtatgcgcgaactgctgtggcgtaaagaagattgccagcgtttttatcgcgaacatgaaggccgtttcttttatcaacgcctggttgaattcatggcctctggtccgattcgcgcatatatcctggctcacaaagatgcgattcagctgtggcgtaccctgatgggtccgacgcgcgtctttcgtgcacgtcatgtggcaccggactcaatccgtggctcgttcggtctgaccgatacgcgcaataccacgcacggtagcgactctgttgttagtgcgtcccgtgaaatcgcggcctttttcccggacttctccgaacagcgttggtacgaagaagaagaaccgcaactgcgctgtggcccggtctgttattctccggaaggtggtgtccattatgtggcgggcacgggtggtctgggtccggcatga (SEQID NO: 78)

(amino acid)

MLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPVCYSPEGG

Like the new mutants, NME6 and NME7 can be expressed with arbitrary affinity tags, but with the following tags:

Histidine tag

(ctcgag) caccaccaccaccaccactga (SEQ ID NO: 84)

Strept II tag

(accggt) tggagccatcctcagttcgaaaagtaatga (SEQID NO: 85)

Example 6-Human NME7-1 sequence optimized for E. coli expression

NME7 wt-cDNA, a codon optimized for expression in E. coli, was generated by Genscript (NJ) at the request of the present inventor. NME7-1 was amplified by the polymerase chain reaction (PCR) using the following primers:
Forward 5'-atcgatcatatgaatcactccgaacgc-3'(SEQ ID NO: 141)
Reverse 5'-agagcctcgagattatccagaattttgaaaaagtattg-3'(SEQ ID NO: 142)

The fragments were then purified, digested (NdeI, XhoI) and cloned between the restriction sites NdeI and XhoI of the expression vector pET21b.

Example 7-Human NME7-2 sequence optimized for E. coli expression

NME7-2 was amplified by the polymerase chain reaction (PCR) using the following primers:
Forward 5'to-atcgatcatatgcatgacgttaaaaatcac-3'(SEQ ID NO: 143)
Reverse 5'-agagcctcgagattatccagaattttgaaaaagtattg-3'(SEQ ID NO: 144)

The fragments were then purified, digested (NdeI, XhoI) and cloned between the restriction sites NdeI and XhoI of the expression vector pET21b.

Example 8-Human NME7-A sequence optimized for E. coli expression

NME7-A was amplified by the polymerase chain reaction (PCR) using the following primers:
Forward 5'-atcgacatatggaaaaaacgctggccctgattaaaccggatg-3'(SEQID NO: 145)
Reverse 5'-actgcctcgaggaaaaacagttccatttcacgagctgccgatg-3'(SEQID NO: 146)

The fragments were then purified, digested (NdeI, XhoI) and cloned between the restriction sites NdeI and XhoI of the expression vector pET21b.

Example 9-Human NME7-AB sequence optimized for E. coli expression
NME7-AB was amplified by the polymerase chain reaction (PCR) using the following primers:
Forward 5'-atcgacatatggaaaaaacgctggccctgattaaaccggatg-3'(SEQID NO: 147)
Reverse 5'-agagcctcgagattatccagaattttgaaaaagtattg-3'(SEQ ID NO: 148)

The fragments were then purified, digested (NdeI, XhoI) and cloned between the restriction sites NdeI and XhoI of the expression vector pET21b.
The protein is expressed on the C-Term His Tag.

NME7-AB was amplified by the polymerase chain reaction (PCR) using the following primers:
Forward 5'-atcgacatatggaaaaaacgctggccctgattaaaccggatg-3'(SEQID NO: 149)
Reverse 5'-agagcaccggtattatccagaattttgaaaaagtattg-3'(SEQ ID NO: 150)

The fragments are then purified, digested (NdeI, Agel) and cloned between the restriction sites NdeI and Agel of the expression vector pET21b, where XhoI is replaced by two stop codons prior to AgeI and His Tag following Strep Tag II. Was done. The protein is expressed with C-Term Strep Tag II.

Example 10-Human NME6 sequence optimized for E. coli expression:

NME6 was amplified by the polymerase chain reaction (PCR) using the following primers:
Forward 5'-atcgacatatgacgcaaaatctgggctcggaaatg-3'(SEQID NO: 151)
Reverse 5'-actgcctcgagtgccggacccagaccacccgtgc-3'(SEQ ID NO: 152)

The fragments were then purified, digested (NdeI, XhoI) and cloned between the restriction sites NdeI and XhoI of the expression vector pET21b. The protein is expressed with C-Term His Tag.

NME6 was amplified by the polymerase chain reaction (PCR) using the following primers:
Forward 5'-atcgacatatgacgcaaaatctgggctcggaaatg-3'(SEQ ID NO: 153)
Reverse 5'-actgcaccggttgccggacccagaccacccgtgcg-3'(SEQ ID NO: 154)

The fragments are then purified, digested (NdeI, Agel) and cloned between the restriction sites NdeI and Agel of the expression vector pET21b, where XhoI is replaced by two stop codons prior to AgeI and His Tag following Strep Tag II. Was done. The protein is expressed with C-Term Strep Tag II.

Example 11-Generation of recombinant NME7-AB

LB cultures (Luria-Bertani cultures) were planted at 1/10 of the overnight culture and cultured at 37 ° C until OD600 reached ~ 0.5. At this point, recombinant protein expression was induced with 0.4 mM isopropyl-β-D-thio-galactoside (IPTG, Gold Biotechnology), and culture was stopped after 5 hours. After collecting the cells by centrifugation (6000 rpm for 10 minutes at 4 ° C.), the cell pellet was resuspended in running buffer (PBS pH 7.4, 360 mM NaCl and 80 mM imidazole). Then lysozyme (1 mg / mL, sigma), MgCl2 (0.5 mM) and DNAse (0.5 μg / mL, sigma) were added.
Cell suspensions were incubated at 37 ° C. for 30 minutes on a turntable (275 rpm) and sonicated on ice for 5 minutes. Insoluble cell debris was removed by centrifugation (4 ° C, 30 minutes, 20000 rpm). The clear lysate was then applied to a Ni-NTA column (Qiagen) equilibrated with running buffer. The column was washed with running buffer (4CV) supplemented with 30 mM imidazole, and the elution was with running buffer (6CV) supplemented with 70 mM imidazole, followed by a running buffer supplemented with 490 mM imidazole. The protein was eluted from the column with. NME7-AB was further purified by molecular sieving chromatography (Superdex 200) "FPLC".

Example 12-Generation of recombinant NME6

LB cultures (Luria-Bertani cultures) were planted at 1/10 of the overnight culture and cultured at 37 ° C until OD600 reached ~ 0.5. At this point, recombinant protein expression was induced with 0.4 mM isopropyl-β-D-thio-galactoside (IPTG, Gold Biotechnology), and culture was stopped after 5 hours. After collecting the cells by centrifugation (6000 rpm for 10 minutes at 4 ° C.), the cell pellet was resuspended in running buffer (PBS pH 7.4, 360 mM NaCl and 80 mM imidazole). Then lysozyme (1 mg / mL, sigma), MgCl2 (0.5 mM) and DNAse (0.5 μg / mL, sigma) were added. Cell suspensions were incubated at 37 ° C. for 30 minutes on a turntable (275 rpm) and sonicated on ice for 5 minutes. Insoluble cell debris was removed by centrifugation (4 ° C, 30 minutes, 20000 rpm). The clear lysate was then applied to a Ni-NTA column (Qiagen) equilibrated with running buffer. The column was washed with 8 CV and the protein was eluted from the column with running buffer (6 CV) supplemented with 420 mM imidazole. NME6 was further purified by molecular sieving chromatography (Superdex 200) "FPLC".

Example 13-Quantitative PCR analysis of naive and primed genes.

Standard analytical methods were used to perform RT-PCR. The primers used are listed below: RNA was isolated ^{using Trizol Registered Trademark} Reagent (Invitrogen). The cDNA was reverse transcribed with Invitrogren using SuperScript II (Invitrogen), followed by Applied Biosystems gene expression analysis (OCT4 P / N Hs00999634_gH, Nanog P / NHs02387400_g1, KLF2) on the Applied Biosystems 7500 real-time device. P / N Hs00360439_g1, KLF4 P / N Hs00358836_m1, FOXa2 P / NHs00232764_m1, OTX2 P / N Hs00222238_m1, LHX2 P / N Hs00180351_m1, XIST P / NHs01079824_m1 and GAPDH P / N 4310884E) , NANOG and OCT4 were analyzed. Each sample was run repeatedly 3 times. Gene expression was standardized to GAPDH. The data was represented to the control as a fold change.

Example 14-MUC1 pull-down analysis shows that NME1, NME6 and NME7 bind to MUC1 protein.

A pull-down analysis was performed on the cell panel using an antibody against the MUC1 * cytoplasmic tail (Ab-5). Proteins pulled down by MUC1 antibody were separated by SDS-PAGE and tested with antibodies specific for NME1, NME6 and NME7 using Western blot techniques. MUC1 * positive breast cancer cell line T47D cell (ATCC), human embryonic stem cell line BGO1v (Life Technologies), human ES cell (HES-3, BioTime Inc.) and human iPS cell (SC101A-1, System Biosciences Inc.)) T47D Cancer cells were grown by the ATCC protocol at RPMI-1640 (ATCC) + 10% FBS (VWR). All stem cells were cultured in the smallest stem cell medium "MM" containing 8nM NM23-RS (recombinant NME1 S120G dimer). Stem cells were grown in plastic containers covered with 12.5 μg / mL anti-MUC1 * C3 mab. Cells were lysed on ice for 10 minutes in 200 μL RIPA buffer. After removal of cell debris by centrifugation, the supernatant was used for coimmune precipitation analysis. MUC1 * was pulled down using an Ab-5 antibody (anti-MUC-1 Ab-5, Thermo Scientific) that recognizes the cytoplasmic tail of MUC1 bound to Dynabeads protein G (Life Technologies). Beds were washed twice with RIPA buffer and resuspended in reducing buffer. The supernatant sample was exposed to reducing SDS-PAGE and then the protein was transferred to a PVDF thin film.
The thin film was then inspected with:
A) Anti-NM23-H1 (NME1) antibody (C-20, Santa Cruz Biotechnology);
B) Anti-NME6 (Abnova); or
C) Anti-NM23-H7 antibody (B-9, Santa Cruz Biotechnology);
D) Staining of NME6 was enhanced using Supersignal (Pierce);
E) Staining of NME7 was enhanced using Supersignal.
After culturing with each secondary antibody bound to HRP, the protein was detected by chemiluminescence. The photographs show that native NME1, NME6 and NME7 are present in MUC1 * -positive breast cancer cells, human ES cells, and human iPS cells, and those bound to those MUC1 *. It should be noted that the number of cells present in the HES-3 pellet was less than that present in the other samples.

Example 15-Recombinant NM23 (S120G mutant H1 dimer), NME7-AB, as well as native NME7, can bind to MUC1 * extracellular domain peptides and induce receptor dimerization.

Gold nanoparticles with a diameter of 30.0 nm were covered with an NTA-SAM surface by Thompson et al. (ACS Appl. Mater. Interfaces, 2011, 3 (8), pp 2979 – 2987). NTA-SAM covering the gold nanoparticles was then activated to an equal volume of 180 μM NiSO4, incubated for 10 minutes at room temperature, washed and resuspended to 10 mM phosphate buffer (pH 7.4). The gold nanoparticles were then loaded with the PSMGFR N-10 peptide (QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAHHHHHH (SEQ ID NO: 155)) at a final concentration of 0.5 μM and incubated for 10 minutes at room temperature. The recombinant NME7-AB protein expressed and purified from E. coli was added to the solution free of charge at the indicated concentrations. The particle solution color changes from pink / red to purple / blue when the proteins immobilized on the particles bind to each other or to two different peptides on two different particles at the same time. If the protein added free to the solution causes particle aggregation, it is because binding to a single peptide does not cause two or more particles to be adjacent to each other. This is strong evidence that it dimerizes peptides of the same strain.

Figure 7 (A) shows NTA-Ni-SAM coated nanoparticles loaded with PSMGFR N-10 peptide. NME7-AB was added to the solution free of charge at the indicated concentrations. The change in solution color from pink to purple / blue of the particle assembly indicates the binding between solution-free NME7 and the MUC1 * peptide on the particles. This result indicates that NME7 in solution has two binding sites for the MUC1 * peptide. The anti-MUC1 * antibody Fab completely inhibits the binding, indicating that particle aggregation is due to the specific interaction of the MUC1 * peptide with NME7. (B) shows NME7-AB added freely to the solution at a wide range of concentrations. Particle agglutination is observed, indicating that NME7 can bind to two peptides simultaneously. (C) shows all the proteins added in solution. NME7-AB turned purple almost immediately. NM23-RS (H1 dimer) also began to turn purple almost immediately. The T47D breast cancer cell line lysate, which contains the natural NME7, turned even more prominently purple.

Example 16-Human ES and iPS cells cultured in NME1 dimer or NME7 are in a naive state as evidenced by the lack of condensed histone-3 in the nucleus showing X inactivation (proof of primed state). It is in.

Human ES (HES-3 stem cells: Biotime) and iPS (SC101A-Ipsc: System Biosciences) cells are of NME1 dimer (NM23-RS) or NME7 (NME7-AB construct) at 8-10 passages. Either was added and cultured in minimal medium (MM). Cells were planted on ^{a Vita TM} plate (Thermo Fisher) covered with 12.5 μg / mL of anti-MUC1 * monoclonal antibody (MN-C3) that binds to the distal portion of the PSMGFR sequence of the MUC1 * receptor. Periodically, through 10 passages, stem cell samples are analyzed by immunocytochemistry (ICC) and analyzed on a confocal microscope (Zeiss LSM 510 confocal microscope) to determine cell localization of histone-3. Was done. When histone-3 was condensed in the nucleus (appearing as a single dot), the copy of the X chromosome was inactivated and the cell was no longer in a pure ground or naive state.
If the stem cells return from the primed state (all commercially available stem cells were induced to the primed state by culturing in FGF), histone-3 is considered a "crowd" cloud "". Or it may just be a spot or undetectable.
Figure 12 shows that all control cells from the same source are 100% in primed state rather than naive state, except that they were raised in FGF on MEF by standard protocol. Is shown for histone-3 (H3K27me3) condensed in the nucleus.
Conversely, the same source cells cultured in NME7 during the 10th passage are mostly stem cells without condensed histone-3, indicating that they are pre-X inactivated and truly naive. Had. The insert shown in Figure 12 is one of many isolated clones that are 100% naive.

Example 17-Detection of NME7 in embryonic stem cells and iPS cells

Human ES cells (BGO1v and HES-3), as well as iPS cells (SC101-A1), were cultured in NME-based medium, where the cells were implanted on a layer of anti-MUC1 * antibody. To identify 7 NME species, cells were harvested and lysed in RIPA buffer (Pierce) supplemented with a protease inhibitor (Pierce). Cell lysates (20 μL) were separated by electrophoresis on a 12% SDS-PAGE reducing gel and transferred to PVDF membranes (GE Healthcare). The blot was blocked with PBS-T containing 3% milk and then incubated overnight at 4 ° C. with the primary antibody (anti NM23-H7 clone B-9: Santa Cruz Biotechnology). After washing with PBS-T, the membrane was incubated with horseradish peroxidase (HRP) -linked secondary antibody (goat anti-mouse, Pierce) for 1 hour at room temperature. The signal was detected with the Immuno-Star Chemiluminescence kit (Bio-Rad). Western blots show that NME7 is present as ~ 40 kDa species as well as ~ 25-33 kDa low molecular weight NME7 species, which can be post-translational modifications such as cleavage or alternative splice isoforms.

Example 18 – Detection of NME7 in iPS conditioned medium

iPS conditioned medium (20 μL) was separated by either 12% SDS-PAGE reducing gel electrophoresis and transferred to PVDF membrane (GE Healthcare). The blot was blocked with PBS-T containing 3% milk and then incubated overnight at 4 ° C. with the primary antibody (anti NM23-H7 clone B-9: Santa Cruz Biotechnology). After washing with PBS-T, the membrane was incubated with horseradish peroxidase (HRP) -linked secondary antibody (goat anti-mouse, Pierce) for 1 hour at room temperature. The signal was detected with the Immuno-Star Chemiluminescence kit (Bio-Rad). Western blots show 7 secreted NMEs with a molecular weight of approximately 30 kDa. Note that recombinant NME7-AB has a molecular weight of 33 kDa, is capable of binding to two MUC1 * peptides at the same time, and supports pluripotent stem cell growth, induction of pluripotency and inhibition of differentiation. .. Seven NME species of ~ 25-30 kDa can be post-translational modifications such as cleavage or alternative splice isoforms that can allow secretion from cells.

Example 19-NME7 Immunoprecipitation and analysis by mass spectrophotometry

Pull-down analysis was performed using an NME7-specific antibody (NM23 H7 B9: Santa Cruz) on a panel of MUC1 * -positive cells. Breast cancer cells (T47D) as well as human ES (BGO1v and HES-3) and iPS (SC101-A1) cells were cultured by standard protocol (T47D) or in NME-based medium on the surface of anti-MUC1 * antibodies. It was cultured. Cells were lysed with RIPA buffer (Pierce) supplemented with a protease inhibitor (Pierce). Cell lysates were supplemented with 10 μg of recombinant NME7-AB incubated at 4 ° C for 2 hours. NME7 was then immunoprecipitated overnight at 4 ° C with anti NM23-H7 (B-9, Santa Cruz Biotechnology) bound to Dynabeads protein G (Life technologies).
Beds were washed twice with PBS and immunoprecipitated proteins were electrophoresed on a 12% SDS-PAGE reducing gel. Proteins were detected by silver staining (Pierce). From T47D samples and BGO1v cells, the ~ 23 kDa band of the protein co-immune-precipitated with NME7 was excised and analyzed by Mass Spec (Taplin Mass Spectrometry Facility, Harvard Medical School). Mass spec analysis showed that all resected protein bands contained sequences from the NME7 NDPK A domain as shown below. The underlined sequence of the A domain of NME7 was identified by mass spec.

MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEK TLALIKPDAISKAGEIIEIINK AGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKR LLGPANSGVAR TDASESIR ALFGTDGIRNAAHGPDSFASAAR EMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN ( SEQID NO: 156)

An immunoprecipitated, higher molecular weight protein band with NME7, ~ 30 kDa, was not analyzed by mass specs, it may be a degradation product, an endogenous NME7 protein, or a compatible splice isoform, or cell lysate. It could be NME7-AB to 33 kDa added.

Example 20-ELISA analysis showing that NME7-AB binds to two MUC1 * extracellular domain peptides simultaneously

The PSMGFR peptide carrying C-terminal cysteine (PSMGFR-Cys) was covalently attached to BSA using the Imject Maleimide Activated BSA Kit (Thermo Fisher). PSMGFR-Cys-conjugated BSA was diluted to 10 μg / mL at 0.1 M carbonate / sodium bicarbonate buffer pH 9.6 and 50 μL was added to each well of the 96-well plate. After overnight incubation at 4 ° C., the plates were washed twice with PBS-T and a 3% BSA solution was added to block the remaining binding sites on the wells. After 1 hour at RT, the plates were washed twice with PBS-T and NME7 diluted with PBS-T + 1% BSA was added in different concentrations. After 1 hour at RT, the plates were washed 3x with PBS-T and anti-NM23-H7 (B-9, Santa Cruz Biotechnology) diluted with PBS-T + 1% BSA was 1/500. Added by dilution. After 1 hour at RT, the plates were washed 3x with PBS-T and goat anti-mouse HRP diluted with PBS-T + 1% BSA was added at a dilution of 1/3333. After 1 hour at RT, the plates were washed 3x with PBS-T and NME7 binding was analyzed at 415 nm using ABTS solution (Pierce).

ELISA MUC1 * Dimerization:
The protocol for NME7 binding was used, and NME7 was used at 11.6 μg / mL.

After 1 hour at RT, the plates were washed 3x with PBS-T and differed in HisTagged PSMGFR peptide (PSMGFR-His) or biotinylated PSMGFR peptide (PSMGFR biotin) diluted with PBS-T + 1% BSA. Added in concentration. After 1 hour at RT, the plates were washed 3x with PBS-T and anti-Histag HRP (Abcam) or streptavidin-HRP (Pierce) diluted with PBS-T + 1% BSA at a concentration of 1/5000. Added. After 1 hour at RT, the plate was washed 3x with PBS-T and was already bound to another PSMGFR peptide that bound BSA, which could not be signaled by anti-His antibody, or streptavidin. Binding of PSMGFR peptide to NME7 was analyzed at 415 nm using ABTS solution (Pierce).

Example 21-NME6 cloning, expression and purification

WT NME6 cDNA, a codon optimized for expression in E. coli, was synthesized by Genscript, NJ at the request of the present inventor. The WT NME6 cDNA was then amplified by the polymerase chain reaction (PCR) using the following primers:
5'-atcgacatatgacgcaaaatctgggctcggaaatg-3'(SEQ ID NO: 157) and
5'-actgcctcgagtgccggacccagaccacccgtgc-3'(SEQID NO: 158).
After digestion with NdeI and XhoI restriction enzymes (New England Biolabs), the purified fragments were cloned into pET21b vectors (Novagen) digested with the same restriction enzymes.

Example 22-NME6 protein expression / purification

LB broth (Luria-Bertani broth) was planted at 1/10 of the overnight culture and cultured at 37 ° C. until OD600 reached ~ 0.5. At this point, recombinant protein expression was induced with 0.4 mM isopropyl-β-D-thio-galactoside (IPTG, Gold Biotechnology), and culture was stopped after 5 hours. After harvesting the cells by centrifugation (4 ° C., 10 minutes, 6000 rpm), the cell pellet was resuspended in running buffer (PBS pH 7.4, 360 mM NaCl, 10 mM imidazole and 8 M urea). Cell suspensions were incubated at 37 ° C. for 30 minutes on a rotating platform (275 rpm) and sonicated on ice for 5 minutes. Insoluble cell debris was removed by centrifugation (4 ° C., 30 minutes, 20000 rpm). The clear lysate was then applied to a Ni-NTA column (Qiagen) equilibrated with running buffer. The column was washed with running buffer 4 CV supplemented with 30 mM imidazole before eluting the protein from the column with running buffer (8 CV) supplemented with running buffer 4 CV and then 420 mM imidazole. The protein was then refolded by dialysis.

Example 23 – Refolding Protocol
1. Overnight dialysis separation for 100 mM Tris pH 8.0, 4 M urea, 0.2 mM imidazole, 0.4 M L-arginine, 1 mM EDTA and 5% glycerin
2. 24-hour dialysis separation for 100 mM Tris pH 8.0, 2 M urea, 0.2 mM imidazole, 0.4 M L-arginine, 1 mM EDTA and 5% glycerin
3. 24-hour dialysis separation for 100 mM Tris pH 8.0, 1 M urea, 0.2 mM imidazole, 0.4 M L-arginine, 1 mM EDTA and 5% glycerin
4. 8-hour dialysis separation for 100 mM Tris pH 8.0, 0.2 mM imidazole, 0.4 M L-arginine, 1 mM EDTA and 5% glycerin
5. Overnight dialysis separation for 25 mM Tris pH 8.0, 0.2 mM imidazole, 0.1 M L-arginine, 1 mM EDTA and 5% glycerin
6. 3 x 3 hour dialysis separation for PBS pH 7.4, 0.2 mM imidazole, 1 mM EDTA and 5% glycerin
7. Overnight dialysis separation for PBS pH 7.4, 0.2 mM imidazole, 1 mM EDTA and 5% glycerin
The refolded protein was centrifuged (18,500 rpm) at 8.4 ° C. for 30 minutes and the supernatant was collected for further purification. In addition, the protein was purified by molecular sieving chromatography (Superdex 200).

All of the references cited herein are carried out by reference.

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One of ordinary skill in the art will be able to recognize or confirm equivalence to each embodiment of the invention described herein using routine experiments. Such equivalents are intended to be included in the claims.

Claims (4)

Agent, SEQ ID NOS: 122-133 is an antibody that recognizes a peptide consisting of any one amino acid sequence selected from agents that inhibit the function of NME7 protein, or
A drug that inhibits the function of the NME 7-AB protein, which is an antibody that recognizes NME 7-AB consisting of the amino acid sequence shown in SEQ ID NO: 39 or SEQ ID NO: 59. The agent according to claim 1, wherein the agent is Fab, monovalent, divalent, or IgM, bispecific human or humanized antibody. The function of the inhibited NME 7 or 7-AB protein is the ability to:
Promotes stem cell proliferation and / or inhibits differentiation;
Promotes cancer cell growth and / or inhibits differentiation;
Combine with MUC1 *;
Binds to DNA;
Acts as a transcriptional regulator;
Secreted by cells; or
The agent according to claim 1, which forms a dimer. Claims for cancer patients or patients at risk of developing cancer, including administering to cancer patients or patients at risk of developing cancer an effective amount of a drug that inhibits the tumorigenic activity of the NME 7 or 7-AB protein Item 2. The agent according to any one of Items 1 to 3.

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